Compounds for medicinal applications

ABSTRACT

The present invention provides compounds for use in treating and/or preventing influenza. The compound comprises a first and second domain in which the first domain comprises at least one anchoring group which binds to the surface of influenza viruses and the second domain comprises at least one anionic group. The first and second domains are covalently linked. Also provided are pharmaceutically acceptable salts, solvates, prodrugs or stereoisomer of the compounds.

FILING DATA

This application is a continuation of PCT/AU2016/051100, filed Nov. 16,2016, which is associated with and claims priority from Australianpatent application no. 2015904895 filed on 20 Nov. 2015 the entirecontents of which is incorporated herein by reference.

FIELD OF INVENTION

This application relates to compounds for medicinal use, in particularfor the treatment or prophylaxis of viral infections. This applicationalso relates to methods for preparing the compounds.

BACKGROUND

Influenza is a respiratory infection contracted by 5% to 25% of the USpopulation annually, roughly 200,000 of whom are hospitalized and 25,000die. Influenza infection continues to be a constant threat to humanhealth and a burden on health services. More recently, the emergence ofhighly pathogenic H5N1 viruses and H7N9 viruses from avian sources, andtheir potential to acquire human transmissibility have increased therisk associated with influenza infection. Although vaccines remain thecornerstone of prevention, significant time is required to develop aneffective vaccine against frequent mutations of any new virus strain.Anti-influenza drugs such as the viral neuraminidase inhibitors (NAinhibitors), including Zanamivir (Relenza) and Oseltamivir (Tamiflu) andM2 ion-channel blockers derived from adamantanes, including amantidineand rimantidine, are available and have been marketed for over 20 years,but their effectiveness is limited by the mechanism of action. Theireffectiveness can be further compromised by viral mutation anddevelopment of subsequent drug resistance.

For example, M2 ion-channel blockers are currently not recommended foruse because almost all circulating season A virus (includingH1Nipcim09), carry a S31N point mutation in the M2 gene that impartsresistance to both amantadine and rimantadine. This has resulted in aheavy reliance on alternative NA inhibitors such as Zanamivir,Oseltamivir and Peramivir.

The NA inhibitors affect the assembly and budding of viruses ofInfluenza A in the viral life cycle. For the progeny virions to bereleased from the cell, neuraminidase (NA) must cleave the sialic acidgroup from the host glycoproteins, which is essential for viral spreadand reinfection. Thus, blocking the function of neuraminidase withspecific inhibitors (NA inhibitors) is a way of treating influenza.However, as such inhibition occurs in the later stage of the viral lifecycle (that is, after viral entry, replication, assembly, and buddingonto cell surface), much of the damage has already been done to thecell. Moreover, only the budding viruses on the cell surface have beenaffected. The uninhibited viral progeny may infect new cells. This leadsto the progression of disease severity. In order to reduce severity ofthe disease, the optimum therapeutic window for treatment with NAinhibitor drugs is early in the disease progression. However, as thetherapeutic efficacy of a given active agent is generally determined bythe mechanism of action, NA inhibitor drugs are considered to berelatively mild and of limited clinical benefit.

There is a need for new approaches to the treatment of influenza.

The life cycle of Influenza A comprises several steps:

(a) Influenza A virus has a lipid bilayer envelope, within which areeight RNA genomic segments, each of which is associated with thetrimeric viral RNA polymerase (PB1, PB2, PA) and coated with multiplenucleoproteins (NPs) to form the vRNPs. The outer layer of the lipidenvelope is spiked with multiple copies of HA, NA and a small number ofM2, whereas the M1 molecules keep vRNPs attached to the inner layer.

(b) The viral surface glycoprotein HA binds to the host cell-surfacesialic acid receptors, and the virus is transported into the cell in anendocytic vesicle. The low pH in the endosome triggers a conformationalchange in the HA protein that leads to fusion of the viral and endosomalmembranes. The low pH also triggers the flow of protons into the virusvia the M2 ion channel, thereby dissociating the vRNPs from M1 matrixproteins. When the M1 molecules are dissociated, the vRNPs that arereleased into the cytoplasm are transported into nucleus by recognitionof the nuclear localization sequences (NLSs) on nucleoproteins.

(c) In the nucleus, the viral polymerase initiated viral mRNA synthesiswith 5′-capped RNA fragments are cleaved from host pre-mRNAs. The PB2subunit binds the 5′-cap of host pre-mRNAs, and the endonuclease domainin the PA subunit cleaves the pre-mRNA 10 to 13 nucleotide downstreamfrom the cap. Viral mRNA transcription is subsequently initiated fromthe cleaved 3′-end of the capped RNA segment. This “cap snatching”occurs on nascent pre-mRNAs.

(d) Viral mRNAs are transported to the cytoplasm for translation intoviral proteins. The surface proteins HA, M2 and NA are processed in theendoplasmic reticulum (ER), glycosylated in the Golgi apparatus andtransported to the cell membrane.

(e) The NS1 protein of the influenza A virus serves a critical role insuppressing the production of host mRNAs by inhibiting the 3′-endprocessing of host pre-mRNAs, consequently blocking the production ofhost mRNAs, including interferon-β mRNAs. Unlike host pre-mRNAs, theviral mRNAs do not require 3′-end processing by the host cell machinery.Therefore, the viral mRNAs are transported to the cytoplasm, whereas thehost mRNA synthesis is predominantly blocked.

(f) The viral polymerase is responsible for not only capped RNA-primedmRNA synthesis but also unprimed replication of vRNAs as follows:

-   -   (−) vRNA→(+) cRNA→(−) vRNA.

Nucleoprotein molecules are required for these two steps of replicationand are deposited on the cRNA and vRNA during RNA synthesis. Theresulting vRNPs are subsequently transported to the cytoplasm, mediatedby M1-NS2 complex that is bound to the vRNPs; NS2 interacts with humanCRM1 protein which export the vRNPs from the nucleus.

(g) The vRNPs reach the cell membrane to be incorporated into newviruses that are budded out. The HA and NA proteins in new virusescontain terminal sialic acids that would cause the viruses to clumptogether and adhere to the cell surface. The NA of newly formed virusescleaves these sialic acid residues, thereby releasing the virus from thehost cell.

During steps (a) and (b) of the influenza viral life cycle, the viralsurface glycoprotein Haemagglutinin (HA) binds to sialic acid receptorson the host cell, followed by receptor-mediated endocytosis of the virusinto the cell. The low pH in the endosome triggers a conformationalchange in the HA protein, that leads to fusion of the viral andendosomal membrane. Meanwhile, the low pH also triggers the flow ofprotons into the virus via the M2 ion channel, thereby releasing ofviral genetic material to the nucleus to start the viral infectionprocess in the cell.

It is recognised that the exposure of virus to low pH in theextracellular environment inactivates the virus. Low pH intranasal gelsprays have been shown to suppress influenza viruses in vitro andprotect ferrets against influenza infection. However, in such studiesthe effective concentration of the acids are around 0.15M (approx. pH3.5). Furthermore, there needs to be contact with the virus to have aviracidal effect. Additionally, the sprays have demonstrated efficacyonly during the early stages of viral infection. Although a pH valueclose to 3.5 can be achieved by administration of a pH 3.5 bufferednasal spray, there is a limitation of this nasal delivery, whichprovides the relatively short product retention time in the nose due tomucociliary clearance. The normal residence time of nasally administeredsolution in human is around 12 to 15 minutes.

Furthermore, in the case of treatment for viral infection, an acidicbuffer would need to be delivered deep into the air ways. It is likelythat the acidic nature of such a buffer will cause irritation andintolerance in humans. Therefore, acidic intranasal sprays would not besuitable as therapeutic agents for the treatment of influenza virusinfection since they are essentially limited and nonspecific.

Polyanionic compounds offer attractive features as anti-HIV agents. Theantiviral activity spectrum of the polyanions extends to a variety ofenveloped viruses including ortho- and para-myxo viruses (influenzavirus). Such polyanions have been shown to inhibit viral replication incell cultures at a concentration of 0.1 to 1 μg/mL. Advantageously, suchcompounds are not cytotoxic even at concentrations up to 10,000 foldhigher. However, these substances suffer from a number ofpharmacokinetic and toxicological drawbacks which seem to compromisetheir clinical utility, for example, in bloodstream they may be retainedby various plasma proteins before reaching their site of action.Additionally, some sulphated polysaccharides are notorious for undesiredanticoagulant activity.

There is a need for new antiviral agents which are effective againstinfluenza. In particular, there is a need for new antiviral agents whichexhibit one or more of the following: i) high potency against influenzaviruses, ii) fast action to control the infection, iii) broad spectrumantiviral activity (including influenza A, B, avian flu), iv) efficacyagainst drug resistant strains, v) low tendency of drug resistance, vi)extended duration of efficacy, vii) high selectivity, viii) improvedtherapeutic window; ix) low toxicity, x) fewer side effects and/or xi)are suitable for therapeutic and prophylactic application.

SUMMARY

The present invention provides compounds and methods for the treatmentand/or prevention of influenza infection.

The compounds of the present invention, also referred to as “surfaceengineering compounds”, are thought to alter the viral surface,converting the surface into a micro acidic/anionic environment toselectively interfere with the viral life cycle, including steps (a) and(b) outlined above.

This is believed to be achieved by the compounds of the presentinvention which combine a group which binds to the influenza virus andwith an acidic or anionic group(s) which act to interfere with thebinding of the haemagglutinin to the cell. This provides an advantage asthe compounds of the invention act to prevent infection of the cell andexerts its effect extracellularly. This avoids the need for the moleculeto invade the cell which should result in lower toxicity and/or fewerside effects.

In one aspect, the present invention provides a compound comprising afirst and second domain,

-   -   the first domain comprising at least one anchoring group which        binds to the surface of influenza viruses; and    -   a second domain comprising at least one anionic group;    -   or a pharmaceutically acceptable salt, solvate, prodrug or        stereoisomer thereof, wherein the first and second domains are        covalently linked.

In another aspect, there is provided compounds wherein the first andsecond domains are covalently linked via at least one divalent linker.

In still other aspects, the compounds of the invention may furthercomprise a multivalent backbone group.

In another aspect, there is provided compounds of Formula (I):[A-L¹

_(n)B

L²-C]_(p)   Formula (I)

or a pharmaceutically acceptable salt, solvate, prodrug or stereoisomerthereof,

wherein:

A at each occurrence is an anchoring group;

B is a multivalent backbone group;

C at each occurrence is an anionic group;

L¹ and L² at each occurrence are divalent linkers;

n is an integer from 1 to 4; and

p is an integer from 1 to 3.

In other aspects, there is provided pharmaceutical compositionscomprising a compound of the invention or a pharmaceutically acceptablesalt, solvate, prodrug or stereoisomer thereof. In further aspects,there is provided the use of a compound of the invention in the or apharmaceutically acceptable salt, solvate, prodrug or stereoisomerthereof in the manufacture of a medicament for the treatment orprevention of influenza viral infection.

In other aspects, there is provided methods of treating or preventinginfluenza viral infection, comprising administering to a person in needthereof a therapeutically effective amount of a compound of theinvention or a pharmaceutically acceptable salt, solvate, prodrug orstereoisomer thereof.

In some aspects, the influenza viral infection is influenza A, influenzaB, avian flu or a drug resistant strain of influenza.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs.

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge.

All documents cited herein are each incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Summary of variable conditions for pre-treatment of virus(A/Sydney/250/99 or A/Sydney/5/97) with test compounds prior toinfection of MDCK cells.

FIG. 2: Pre-treatment of virus (A/Sydney/250/99) with test compoundsprior to infection of MDCK cells, A) MD348, B) MD345, and C) Zanamivir(control).

FIG. 3: Pre-treatment of virus (A/Sydney/5/97) with test compounds priorto infection of MDCK cells, A) MD348, B) MD345, and C) Zanamivir(control).

FIG. 4: Pre-treatment of virus (A/Sydney/250/99) with MD345 (testcompound) and Zanamivir (control) over varying time periods andconditions prior to infection of MDCK cells.

FIG. 5: Comparison of antiviral activity of MD314-1 and MD021-7 againstA/Sydney/5/97 (H3N2).

FIG. 6: Comparison of antiviral activity of MD314-1 and MD021-7 againstA/Mississippi/03/01 (H1N1) Wild Type.

FIG. 7: Comparison of antiviral activity of MD021-7 and MD051-3 againstA/Mississippi/03/01 (H1N1) Wild Type.

FIG. 8: Comparison of antiviral activity of MD021-7 and MD051-3 againstA/Mississippi/03/01 (H1N1) H274Y (Oseltamivir resistant).

FIG. 9: Comparison of antiviral activity of MD314-1 and MD021-7 againstA/Mississippi/03/01 (H1N1) H274Y (Oseltamivir resistant).

FIG. 10: Summary of timing of treatments, infection and organ harvestfor assessment of rates of viral clearance in lungs and nasal turbinatesin mice.

FIG. 11: Comparative lung viral titres in mice for assessment of ratesof viral clearance with MD021, Zanamivir and PBS.

FIG. 12: Comparative lung viral titres in mice for assessment of ratesof viral clearance with MD021, Zanamivir and PBS at days 1, 3, 5 and 7post infection.

FIG. 13: Comparative viral titre expressed as a percentage of the meanof the PBS control group.

FIG. 14: Comparative weight loss in mice for assessment of rates ofviral clearance with MD021, Zanamivir and PBS.

FIG. 15: Neutralisation of A/California/7/09 (pan H1N1) in vitro.

DETAILED DESCRIPTION

As discussed above the compounds of the present invention are thought toalter the viral surface, converting the surface into a microacidic/anionic environment to selectively interfere with steps in theviral life cycle.

It is believed that the acidic/anionic groups of the compounds of thepresent invention convert the viral surface into a micro acidic/anionicenvironment which reduces or inactivates viral activity. Furthermore, itis believed that these compounds act extracellularly and may thereforebe associated with reduced incidence of side effects, reduced toxicityand/or a reduction in the tendency for resistance. More particularly, itis believed that the compounds of the present invention actextracellularly at multiple target points of the viral life cycle,including but not limited to:

-   -   Acidic/anionic groups generate a low pH at the viral surface,        which triggers premature conformational change in the        haemagglutinin (HA) protein, which may subsequently reduce or        retard the fusion of the virus and the cells. This is specific        to viral life cycle step (b) referred to above.    -   Additionally, it is believed that the creation of a low pH        environment on the viral surface sparks a flow of protons into        the virus via M2 ion channel, which may cause the release of        genetic material outside of cells. This is specific to viral        life cycle step (b) referred to above.    -   It is believed that the anchor groups of the compounds of the        invention may cross link NA/HA and aggregate the virus particles        to reduce the mobility and infectivity of viruses. This is        specific to viral life cycle steps (a) & (b) referred to above.    -   It is believed that the anchor groups inhibit the function of        neuraminidase (NA) to reduce or prevent the release of new        viruses from the cell membrane. This is specific to viral life        cycle step (g) referred to above.

It is believed that the disruption of viral life cycle steps (a) and (b)may reduce the potential severity of infection by reducing viralinfectivity including viral invasion into cells, viral disruption ofnormal cell nucleus functions, and/or viral replication. Accordingly, itis considered that the administration of the compounds of the presentinvention may provide a more effective treatment of viral infectionand/or faster recovery for patients than currently available NAinhibitor drugs. Further it is believed that the compounds of thepresent invention can used prophylactically in the prevention ofinfluenza infection.

Accordingly, in one aspect, the present invention a compound comprisinga first and second domain,

-   -   the first domain comprising at least one anchoring group which        binds to the surface of influenza viruses; and    -   a second domain comprising at least one anionic group;    -   or a pharmaceutically acceptable salt, solvate, prodrug or        stereoisomer thereof, wherein the first and second domains are        covalently linked.

In some aspect, the first and second domains may be covalently linkedvia at least one divalent linker. In other aspects, the compounds of theinvention may further comprise a multivalent backbone group.

In another aspect, the present invention provides compounds of Formula(I):[A-L¹

_(n)B

L²-C]_(p)   Formula (I)

or a pharmaceutically acceptable salt, solvate, prodrug, ester orstereoisomer thereof,

wherein:

A at each occurrence is an anchoring group;

B is a multivalent backbone group;

C at each occurrence is an anionic group;

L¹ and L² at each occurrence are divalent linkers;

n is an integer from 1 to 4; and

p is an integer from 1 to 4.

As used herein, “a compound of the invention” or “a compound of formula(I)” means a compound or a pharmaceutically acceptable salt, solvate,prodrug, ester or stereoisomer thereof, or a physiologically functionalderivative thereof.

The term “anchoring group” as used herein refers to any suitable groupwhich binds to the surface of influenza viruses. In some embodiments,the anchoring group is a group which binds to neuraminidase, such as aneuraminidase inhibitor or a derivative thereof. Examples of suitablegroups include, but are not limited to, a sialic acid or a derivativethereof, including N- or O-substituted derivatives of neuraminic acidsuch as zanamivir or laninamivir, oseltamivir, peramivir, 2,3-difluorosialic acids (DFSA) and derivatives thereof. In other embodiments, theanchoring group comprises an antibody binding domain which specificallybinds neuraminidase. Examples of suitable anchoring group comprising anantibody binding domain which specifically binds neuraminidase includean antibody (Ab) or an antigen binding fragment of an antibody, a singlechain antibody fragment (scFV) or a single domain antibody (dAb).

The term “backbone group” as used herein refers any suitable multivalentgroup to which one or more anchoring groups and one or more acidic oranionic groups are attached. In some aspects, the backbone group may bea trivalent group. In other aspects, the backbone group may be atetravalent group. Examples of suitable backbone groups include, but arenot limited to, optionally substituted propane-1,2,3-tricarboxylate,optionally substituted cyclohexane-1,3,5-tricarboxylate, and optionallysubstituted benzene-1,2,4,5-tetracarboxylate.

The terms “acidic or anionic group” and “acidic/anionic group” refers toany suitable functional group having acidic and/or anionic propertieswhich acts to interfere with the binding of the haemagglutinin to thecell. Specifically, the term “anionic” describes the net negative chargeof a functional group or material. It will be understood that a givennegatively charged material may have one or more positively chargedcounterions associated with it, or vice versa. In solution, a negativelycharged material may have dissociated from one or more positivelycharged counterions with which it is associated. As used herein, theterm “anionic” is used to describe a property of that material orfunctional group and not the overall complex with one or morecounterions which will typically render the complex neutral. It isunderstood that certain functional groups are negatively charged,neutral or positively charged at varying values of pH. Whether amaterial is anionic will be determined based on the sum of thesecharges. For example, within the scope of the invention acidic oranionic group may include one or more of the following functional groupscarboxylic, sulfonic, phosphoric, and phosphinic acids and theirisosteres or bioisosteres. Examples of suitable acidic or anionic groupsinclude, carboxylic acids, amino acids, dipeptides, tripeptides orpolypeptides or a derivative thereof having acidic and/or anionicproperties, such as aspartic acid, cysteic acid, glutamic acid orpeptides derived from aspartic acid, cysteic acid and/or glutamic acid.

The term “sialic acid” is well understood in the art and refers to a9-carbon monosaccharide, especially those derived from neuraminic acid.Examples of suitable groups include, but are not limited to, optionallysubstituted monosaccharides derived from N- and/or O-substitutions ofN-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc).

The term “neuraminidase inhibitor” is well understood in the art andrefers to a class of compounds which block the neuraminidase enzyme. Insome embodiments, the anchoring group may be a neuraminidase inhibitoror a derivative thereof. Examples of suitable neuraminidase inhibitorsinclude, but are not limited to, a sialic acid or a derivative of asialic acid, such as a 2,3-difluoro sialic acid. Other suitableneuraminidase inhibitors include:

and derivatives thereof.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody with a second chemicalspecies, mean that the interaction is dependent upon the presence of aparticular structure (e.g., an antigenic determinant or epitope) on thechemical species; for example, an antibody recognises and binds to aspecific protein structure rather than to proteins generally.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Non-limitingembodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE,IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG 3, IgG4, IgA1 andIgA2) or subclass.

The term “antigen binding domain” or “antigen binding fragment” of anantibody, as used herein, refers to one or more fragments of an antibodyor protein that retain the ability to specifically bind to an antigen(e.g., IL-12). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody. Suchantibody embodiments may also be bispecific, dual specific, ormulti-specific formats; specifically binding to two or more differentantigens. Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al, 1989 Nature 341 544-6, Winter et al., PCT publication WO90/05144 A1 herein incorporated by reference), which comprises a singlevariable domain; and (vi) an isolated complementarity determining region(CDR). Furthermore, although the two domains of the Fv fragment, VL andVH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); (see e.g., Birdet al, 1988 Science 242 423-6; Huston et al, 1988 Proc Natl Acad Sci USA85 5879-83). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (seee.g., Holliger, P., et al., 1993, Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al., 1994, Structure 2:1121-1123). Suchantibody binding portions are known in the art (Kontermann and Dubeleds., Antibody Engineering 2001 Springer-Verlag. New York. 790 pp., ISBN3-540-41354-5).

In one embodiment, the anchoring group at each occurrence is anysuitable anchoring group which binds to the surface of influenzaviruses. In some embodiments, the anchoring group at each occurrence isa group which binds to neuraminidase, including a neuraminidaseinhibitor or a derivative thereof. In certain embodiments, the anchoringgroup is a neuraminidase inhibitor or a derivative thereof selected froma sialic acid, such as zanamivir or laninamivir, a 2,3-difluoro sialicacid, oseltamivir, peramivir, and derivatives thereof. In otherembodiments, the anchoring group at each occurrence comprises anantibody binding domain which specifically binds neuraminidase. Incertain embodiments where the anchoring group is an antibody bindingdomain which specifically binds neuraminidase, the anchoring group isselected from the group consisting of an antibody (Ab), an antigenbinding fragment of an antibody, a single chain antibody fragment (scFV)and a single domain antibody (dAb). The compounds of the invention maycomprise a total of 1 to 6 anchoring groups, preferably 2 to 4 anchoringgroups, more preferably 2 anchoring groups.

In another embodiment, the anchoring group is a monovalent radicalderived from a group of formula II

a group of formula III

a group of formula IV

and a group of formula V

-   -   wherein E₁ is selected from the group consisting of —COOH,        COOHR₆, —SO₃H, —PO₃H₂, —PO(OR₆)₂    -   G₁ is selected from —NH₂, —CH₂NH₂, —(CH₂)₂NH₂,

-   -   T₁ is selected from the group consisting of an optionally        substituted C₁-C₆ alkyl, optionally substituted C₂-C₆ alkenyl,        or an optionally substituted C₂-C₆ alkynyl    -   U₁ is selected from the group consisting of —CH₂R₇, —(CH₂)₂R₇,        —CH₂CHR₇CH₂R₇    -   R₇ is selected from the group consisting of H, —OH, —OCH₃, —OAc,        —NH₂, or —SH.    -   R₈ and R_(8′) are each independently selected from the group        consisting of hydrogen —N₃, —CN, —CH₂NH₂, guanadino, or        —NR₁₀R₁₁;    -   R₉ is selected from hydrogen, optionally substituted C₁-C₆        alkyl, optionally substituted C₂-C₆ alkenyl, or optionally        substituted C₂-C₆ alkynyl;    -   R₁₀ and R₁₁ are each independently selected from the group        consisting of hydrogen, optionally substituted C₁-C₆ alkyl        group, optionally substituted C₁-C₆ acyl, —C(NH)NH₂, —CH₂COOH,        CH₂CH₂OH or —CH₂CH(R₁₂)(R₁₃),    -   R₁₂ and R₁₃ are each independently selected from O and R₁₄N═,        and    -   R₁₄ is selected from the group consisting of hydrogen, —OH,        —OCH₃, —NH₂, and —N(CH₃)₂,    -   R₁₅ and R_(15′) are each independently selected from hydrogen        and CO₂R₁₆,    -   R₁₆ is H or optionally substituted C₁-C₆ alkyl;    -   R₁₇ is selected from NH₂ and —NHC(═NH)NH₂;    -   G₂ is

-   -   R₁₈ is selected from the group consisting of hydrogen,        optionally substituted C₁-C₆ alkyl, optionally substituted C₂-C₆        alkenyl, and optionally substituted C₂-C₆ alkynyl.

In still other embodiments, the anchoring group at each occurrence isselected from the group consisting of a group of Formula VI and a groupof Formula VII:

-   -   wherein R¹ is selected from the group consisting of —NR₂,        guandino, —ONR₂, wherein R is H or optionally substituted C₁-C₆        alkyl,    -   R² is selected from the group consisting of H, optionally        substituted C₁-C₆ alkyl, and optionally substituted aryl, and    -   R³ is selected from the group consisting of H, optionally        substituted C₁-C₆ alkyl, optionally substituted aryl, and        optionally C₁-C₆ acyl.

In another embodiment, the anchoring group is a group of Formula (VIa):

In a further embodiment, the anchoring group is a group of Formula(VIIa):

In an embodiment, the linker (such as L¹ and/or L² of Formula I) at eachoccurrence is any suitable divalent linker. In one embodiment, thelinker at each occurrence is independently selected from a bond, anoptionally substituted C₁-C₁₂ alkylene, —C(O)—NR—, —O—C(O)O—NR—,—C═N—O—, —C═N—NR—, —SO₂—NR—, disulfide, —C(O)—NR—(CH₂)_(x)—NR—,—(CH₂)_(x)—NR—, —(CH₂)_(x)—(O—(CH₂)_(y)—)_(z)—, optionallysubstituted-C═N—O—, optionally substituted —C═N—NR-alkylene, optionallysubstituted alkylene sulfonamide, an optionally substituted alkylenedisulfide, wherein R is independently H or C₁-C₆ alkyl and wherein x, yand z are each integers independently selected from 0, 1, 2, 3 and 4.

In one embodiment wherein the anchoring group is a group of Formula (VI)or Formula (VIa), the linker L¹ of Formula (I) is selected from—C(O)—NR—(CH₂)_(x)—NR—, —(CH₂)_(x)—NH— and —(CH₂)_(x)—(O—(CH₂)_(y))_(z),wherein x, y and z are each integers independently selected from 0, 1,2, 3 and 4. In another embodiment wherein the anchoring group is a groupof Formula (VII) or Formula (VIIa), the linker L¹ of Formula (I) is—(CH₂)_(x)—NH—, wherein x is an integer selected from 0, 1, 2, 3 and 4.

In one embodiment, the linker L² of Formula (I) is a bond.

In an embodiment, the backbone group is any suitable multivalent group.In one embodiment, the backbone group is a trivalent backbone group. Inanother embodiment, the backbone group is a tetravalent backbone group.In one embodiment, the backbone group is selected from an optionallysubstituted tricarboxylate group, an optionally substitutedtetracarboxylate group or a multivalent peptide. In other embodiments,the backbone group is selected from optionally substitutedpropane-1,2,3-tricarboxylate, optionally substitutedprop-1-ene-1,2,3-tricarboxylate, optionally substitutedcyclopropane-1,2,3-tricarboxylate, optionally substitutedcyclohexane-1,3,5-tricarboxylate, optionally substitutedbenzene-1,3,5-tricarboxylate, optionally substituted methanetetracarboxylate, optionally substituted 1,2,3,4-butanetetracarboxylate, optionally substitutedethylene-1,1,2,2-tetracarboxylate, such as ethylenediaminetetraaceticacid (EDTA), optionally substitutedcyclohexane-1,2,4,5-tetracarboxylate, and optionally substitutedbenzene-1,2,4,5-tetracarboxylate. In still other embodiments, thebackbone group is selected from optionally substitutedpropane-1,2,3-tricarboxylate, optionally substitutedcyclohexane-1,3,5-tricarboxylate, optionally substitutedbenzene-1,2,4,5-tetracarboxylate, optionally substituted pyromelliticacid and optionally substituted di-pyromellitic acid formed by—C(O)—NH—NH—C(O)— bond(5,5′-(hydrazine-1,2-dicarbonyl)bis(benzene-1,2,4-tricarboxylic acid).In still other embodiments, the backbone group is a multivalent peptide.

In an embodiment, the acidic or anionic group is any suitable functionalgroup having acidic and/or anionic properties which acts to interferewith the binding of the haemagglutinin to the cell. In some embodiments,the acidic or anionic group is any suitable group comprising one or morefunctional groups selected from carboxylic acids, sulfonic acids,phosphoric acids, and phosphinic acids and their isosteres orbioisosteres. In some embodiments, the acidic or anionic group is anysuitable amino acid, a dipeptide, a tripeptide, a polypeptide orderivatives thereof comprising one or more acidic functional groups. Inone embodiment, the acidic or anionic group is aspartic acid, cysteicacid, glutamic acid or peptides derived from aspartic acid, cysteic acidand/or glutamic acid.

In an embodiment, the compounds of the invention comprise a total of 2to 26 acidic/anionic residues, preferable 4 to 10 acidic/anionicresidues. For example, where the acidic or anionic group comprises adicarboxylic acid, it is appreciated that such a moiety comprises twodistinct acidic/anionic residues. By way of example with reference tothe representative examples of compounds of the invention, it is notedthat MD349 comprises a total of nine acidic or anionic residues (fivecarboxylic acid residues and four sulfonic acid residues), MD348comprises a total of seven acidic or anionic residues (four carboxylicacid residues and three sulfonic acid residues), MD345 comprises a totalof eight acidic or anionic residues (one carboxylic acid residues andseven sulfonic acid residues), MD352 comprises a total of nine acidic oranionic residues (two carboxylic acid residues and seven sulfonic acidresidues), MD356 comprises a total of six acidic or anionic residues(two carboxylic acid residues and four sulfonic acid residues), MD357comprises a total of six acidic or anionic residues (two carboxylic acidresidues and four sulfonic acid residues), MD358 comprises a total ofeight acidic or anionic residues (eight carboxylic acid residues), MD359comprises a total of eight acidic or anionic residues (two carboxylicacid residues and six sulfonic acid residues), MD373 comprises a totalof eight acidic or anionic residues (eight carboxylic acid residues),and MD376 comprises a total often acidic or anionic residues (tencarboxylic acid residues).

In another embodiment, the acidic or anionic group is a group of FormulaVIII:

wherein

-   -   R⁴ at each occurrence is independently selected from the group        consisting of —COOH, —SO₃H, —NHCH₂SO₃H, —CONH-Cya, —CONH-Asp,        —CONH-Asp(-NHCH₂SO₃H)_(w), and —CONH-Asp(-NHCH₂PO₃H)_(w),        wherein w is an integer 1 or 2;    -   R⁵ is selected from the group consisting of —OH and —NHCH₂SO₃H    -   u is an integer from 0 to 3;    -   t is an integer from 0 to 3;    -   r is an integer from 0 to 6;    -   v is an integer from 1 to 12.

In an embodiment, n is an integer from 1 to 4. In another embodiment, nis an integer from 1 to 3. In certain embodiments, n is 1. In otherembodiments, n is 2. In still other embodiments, n is 3. In furtherembodiments, n is 4.

In an embodiment, p is an integer from 1 to 4. In another embodiment, pis an integer from 1 to 2. In certain embodiments, p is 1. In otherembodiments, p is 2. In still other embodiments, p is 3. In furtherembodiments, p is 4.

Additionally, or alternatively, in some embodiments the compounds ofFormula (I) may be modified, either before, during, or after assembly,to enhance or modify the biological activity and/or binding affinity forthe influenza virus surface. In an embodiment, where the acidic oranionic groups comprise one or carboxylic acids, including amino acids,the carboxylic acid may be modified by sulfation, phosphorylation,functional group interconversion, and/or attachment of side chains. Forexample, where the acidic or anionic groups comprises a carboxylic acid,the carboxylic acid may be modified to an amide or sulphated amide,including —C(═O)NH(CH₂)_(n)SO₃H. Alternatively, or additionally, wherethe acidic or anionic groups comprise an amino acid or peptide, theC-terminus may be modified to include alternative functional groups,such as conversion of the terminal acid to an amide or sulphated amideincluding —C(═O)NH(CH₂)_(n)SO₃H.

With respect to formula (I) compounds disclosed herein the followingcombinations of any or more of (i) to (vii) are contemplated:

-   -   (i) A is Formula (IIa); or        -   A is Formula (IIIa);    -   (ii) B is propane- 1,2,3-tricarboxylic acid; or        -   B is cyclohexane-1,3,5-tricarboxylate, or        -   B is benzene-1,2,4,5-tetracarboxylate;    -   (iii) L¹ is —C(O)—NH—; or        -   L¹ is —C(O)—NH—(CH₂)₂—NH—; or        -   L¹ is —C(O)—NH—(CH₂)₃—NH—; or        -   L¹ is —C(O)—NH—(CH₂)₄—NH—; or        -   L¹ is —C(O)—NH—(CH₂)₅—NH—; or        -   L¹ is —C(O)—NH—(CH₂)₆—NH—; or        -   L¹ is —C(O)—NH—(CH₂)₇—NH—; or        -   L¹ is —C(O)—NH—(CH₂)₈—NH—; or        -   L¹ is —C(O)—NH—(CH₂)₉—NH—; or        -   L¹ is —C(O)—NH—(CH₂)₁₀—NH—; or        -   L¹ is —C(O)—NH—(CH₂)₁₂—NH—; or        -   L¹ is —(CH₂)₆—NH—; or        -   L¹ is —(CH₂)₈—NH—; or        -   L¹ is —(CH₂)₉—NH—; or        -   L¹ is —(CH₂)₁₀—NH—; or        -   L¹ is —(CH₂)₁₂—NH—;    -   (iv) L² is a bond:    -   (v) C is L-aspartic acid; or        -   C is D-aspartic acid; or        -   C is L-cysteic acid; or        -   C is D-cysteic acid; or        -   C is -(L-Asp)₂-OH; or        -   C is -(D-Asp)₂-OH; or        -   C is -(L-Cya)₂-OH; or        -   C is -(D-Cya)₂-OH; or        -   C is -(L-Asp)₃-OH; or        -   C is -(D-Asp)₃-OH; or        -   C is -(L-Cya)₃-OH; or        -   C is -(D-Cya)₃-OH; or        -   C is -(L-Cya)₇-OH; or        -   C is -(D-Cya)₇-OH; or        -   C is -(L-Asp-L-Cya)₄-OH; or        -   C is -(L-Asp-L-Cya)₄-OH; or        -   C is -[L-Asp(L-Cya)]₃-OH; or        -   C is -[D-Asp(D-Cya)]₃-OH; or        -   C is -[L-Asp(L-Cya)]₄-OH; or        -   C is -[(L-Asp)₄-(L-Cya)₃]-OH; or        -   C is -[(D-Asp)₄-(D-Cya)₃]-OH; or        -   C is -[L-Asp(L-Cya)]₃-OH; or        -   C is -[D-Asp(D-Cya)]₃-OH; or        -   C is -[L-Asp(L-Cya)]₄-OH; or        -   C is -[D-Asp(D-Cya)]₄-OH; or        -   C is L-Asp(NHCH₂SO₃H)₂; or        -   C is D-Asp(NHCH₂SO₃H)₂; or        -   C is -[L-Asp(NHCH₂SO₃)]₃NHCH₂SO₃H; or        -   C is -[D-Asp(NHCH₂SO₃)]₃NHCH₂SO₃H;        -   C is D-aspartic acid; or        -   C is L-cysteic acid; or        -   C is D-cysteic acid; or        -   C is -(L-Asp)₂-OH; or        -   C is -(D-Asp)₂-OH; or        -   C is -(L-Cya)₂-OH; or        -   C is -(D-Cya)₂-OH; or        -   C is -(L-Asp)₃-OH; or        -   C is -(D-Asp)₃-OH; or        -   C is -(L-Cya)₃-OH; or        -   C is -(D-Cya)₃-OH; or        -   C is -(L-Cya)₇-OH; or        -   C is -(D-Cya)₇-OH; or        -   C is -(L-Asp-L-Cya)₄-OH; or        -   C is -(L-Asp-L-Cya)₄-OH; or        -   C is -[L-Asp(L-Cya)]₃-OH; or        -   C is -[D-Asp(D-Cya)]₃-OH; or        -   C is -[L-Asp(L-Cya)]₄-OH; or        -   C is -[(L-Asp)₄-(L-Cya)₃]-OH; or        -   C is -[(D-Asp)₄-(D-Cya)₃]-OH; or        -   C is -[L-Asp(L-Cya)]₃-OH; or        -   C is -[D-Asp(D-Cya)]₃-OH; or        -   C is -[L-Asp(L-Cya)]₄-OH; or        -   C is -[D-Asp(D-Cya)]₄-OH; or        -   C is L-Asp(NHCH₂SO₃H)₂; or        -   C is D-Asp(NHCH₂SO₃H)₂; or        -   C is -[L-Asp(NHCH₂SO₃)]₃—NHCH₂SO₃H; or        -   C is -[D-Asp(NHCH₂SO₃)]₃—NHCH₂SO₃H;    -   (vi) n is 2; or        -   n is 3        -   n is 3    -   (vii) p is 1; or        -   p is 2.        -   p is 2.

For ease or reference, representative examples of compounds of theinvention may be abbreviated. For example, in the context of therepresentative examples, for ease reference, the anchoring group(including A of Formula (I)) and linker (including L₁) of Formula (I)),at each occurrence, may be represented as a single group, referred to asZ_(x) or S_(y). Specifically, where the compounds of the inventioncomprise a group of Formula Z_(x), such groups are sialic acidderivatives in which a functional group (amino or guanidino) replacesthe hydroxyl group at 4-position of sialic acid ring. Such amodification enables the groups of Formula Z_(x), to interact withinfluenza viral neuraminidases, but not with mammalian neuraminidases.This provides an aspect of safety when the compounds of the inventionare administered to mammals including humans. The groups of FormulaS_(y) are linked via the 2-hydroxy group of sialic acid. Specifically athio-glycoside linker group is used in conjunction with the anchoringgroup in Formula S_(y), such as 2-S—(CH₂)_(m) NHR, which resists theenzymatic hydrolysis when compared with equivalent ether linkers, suchas 2-O—(CH₂)_(m) NHR. For ease of reference, the structures of Z_(x), orS_(y) are outlined below:

Formula Z_(x)

Structure Z_(x) Abbreviation X is 0 Z₁ X is 2 Z₃ X is 3 Z_(2b) X is 4Z_(b) X is 5 Z_(p) X iS 6 Z X is 7 Z_(h) X is 8 Z₀ X is 9 Z_(n) X is 10Z₁₀ X is 12 Z₁₂

Formula S_(y)

Structure S_(y) Abbreviation Y is 6 S₆ Y is 8 S₈ Y is 9 S₉ Y is 10 S₁₀ Yis 12 S₁₂

For ease or reference, in the context of the representative examples,the compounds from which the backbone group B of Formula (I) may bederived are abbreviated as follows

Abbre- viation Structure Name CHTCA

cis-1,3,5- cyclohexanetricarboxylic acid TCA

Tricarboxylic acid PYR

Pyromellitic acid PYR-NH- Di-pyromellitic acid with a NH-PYRC(O)—NH—NH—C(O) bond 5,5′-(hydrazine-1,2- dicarbonyl)bis(benzene-1,2,4-tricarboxyllc acid)

For ease or reference, in the context of the representative examples,the anchoring group A, linker L¹ and backbone group B of Formula (I),may be abbreviated as follows:

Abbreviation Structure Abbreviation Structure PK2 TCA(Z_(x))₂

PK1 CA(Z_(x))₂

PK1 PYR(Z_(x))₂

PK2 PYR(Z_(x))₂

CHTCA(Z_(x))₂

CHTCA(S_(y))₂

PK2 TCA(S_(y))₂

PK1 TCA(S_(y))₂

PK1 PYR(S_(y))₂

PK2 PYR(S_(y))₂

CHTCA(Z_(x))(S_(y))

CHTCA(Z_(x))₂(S_(y)) PK1 PYR(Z_(x))₂(S_(y))

PK2 PYR(Z_(x))₂(S_(y))

PK2PYR(Z_(x))₂(NHCH₂SO₃H)₂

For ease or reference, in the context of the representative examples,the compounds from which the acidic or anionic groups of Formula (I) maybe derived, are abbreviated as follows:

Abbreviation Structure Name Cya Cya-SO₃H

Cysteic acid L-Cya L-Cya-SO₃H

L-Cysteic acid D-Cya D-Cya-SO₃H

D-Cysteic acid Asp Aspartic acid L-Asp

L-Aspartic acid D-Asp

D-Aspartic acid (L-Asp)₃ or L-Asp-L-Asp-L-Asp

(D-Asp)₃ or D-Asp-D-Asp-D-Asp

(L-Cya)₃ or L-Cya-L-Cya-L-Cya

(D-Cya)₃ or D-Cya-D-Cya-D-Cya

(L-Cya)₇-OH or (L-Cya)₇

(D-Cya)₇-OH (D-Cya)₇

L-Asp(L-Cya)-L- Asp(L-Cya)-L-Asp(L- Cya)-OH or [L-Asp(L-Cya)]₃

D-Asp(D-Cya)-D- Asp(D-Cya)-D- Asp(D-Cya)-OH or [D-Asp(D-Cya)]₃

Asp(NHCH₂SO₃H)- Asp(NHCH₂SO₃H)- Asp(NHCH₂SO₃H)₂

L-(Asp)(β)-L- (Asp)(β)-L-(Asp)

D-(Asp)(β)-D- (Asp)(β)-D-(Asp)

L-(Asp)(β)-D- (Asp)(β)-L-(Asp)

D-Asp(L-Asp)-D- Asp(L-Asp)-D-Asp(L- Asp)

Representative examples of compounds of the invention are outlinedbelow.

Ref. Abbreviated No. Ref. Structure MD021 Mol. Weight. 1243 CHTCA(Z)₂-L-Asp

M0154 Mol. Weight. 1485 CHTCA(Z₀)₂-L- Asp(NHCH₂SO₃H)₂

M0155 Mol. Weight. 1485 CHTCA(Z₀)₂-D- Asp(NHCH₂SO₃H)₂

MD012 Mol. Weight. 1534 CHTCA(Z)₂(S₆)

MD051 Mol. Weight. 1318 PK2 TCA(Z)₂-[L- Asp-L-Asp]

MD185 Mol. Weight. 1852 PK2PYR(Z_(n))₂[(D- Asp)(NHCH₂SO₃)₂]₂

MD314 Mol. Weight. 1982 CHTCA(Z₀)-L- Asp(L-Cya)-L- Asp-(L-Cya)-L-Asp(L-Cya)

MD317 Mol. Weight. 1318 PK2 TCA(Z)₂-[D- Asp-D-Asp]

MD342 Mol. Weight. 2208 PK2 TCA(Z₀)₂- [D-Asp-D-Cya]₄- OH

MD343 Mol. Weight. 1597 PK2 TCA(Z₀)₂- [D-Cya]₃-OH

MD344 Mol. Weight. 2058 PK2 TCA(Z₀)₂[(D- Asp)₄D-Cya)₃]- OH

MD345 Mol. Weight. 2201 PK2TCA(Z₀)₂- [(D-Cya (SO₃H)]₇-OH

MD348 Mol. Weight. 1982 CHTCA(Z₀)₂-[D- Asp(D-Cya)-D- Asp(D-Cya)-D-Asp(D-Cya)]-OH

MD349 Mol. Weight. 2248 CHTCA(Z₀)[D- Asp(D-Cya)-D- Asp(D-Cya)-D-Asp(D-Cya)-D- Asp(D-Cya)]-OH

MD352 Mol. Weight. 1994 PK2 PYR(Z_(n))₂[D- Cya]₇COOH

MD356 Mol. Weight. 1940 PK1 PYR(Z_(n))₂- [D-Cya-D-Cya]₂

MD357 Mol. Weight. 1854 PK2 PYR(Z_(n))₂- D-Cya-D-Cya]₂

MD358 Mol. Weight. 1940 PK1 PYR(Z_(n))₂[L- Asp-L-Asp-L- Asp]₂

MD359 Mol. Weight. 2158 PK1 PYR(Z_(n))₂[(D- Cya)₃-OH]₂

MD371 Mol. Weight. 1710 PK1 PYR(Z_(n))₂- [(D-Asp-D-Asp]₂

MD373 Mol. Weight. 1940 PK1 PYR(Z_(n))₂- [D-Asp-D-Asp- D-Asp]₂

MD376 Mol. Weight. 2170 PK1 PYR(Z_(n))₂- [D-Asp-D-Asp- D-Asp-D-Asp]₂

The term “alkyl” as used alone or in combination herein refers to astraight or branched chain saturated hydrocarbon group. The term “C1-12alkyl” refers to such a group containing from one to twelve carbon atomsand “lower alkyl” refers to C1-6 alkyl groups containing from one to sixcarbon atoms, such as methyl (“Me”), ethyl (“Et”), n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl and the like.

The term “cycloalkyl” refers to non-aromatic, saturated non-aromaticcarbocycles. The term “C4-9 cycloalkyl”, for instance, refers to such agroup having from 4 to 9 carbon atoms. Examples include cyclobutyl,cyclopentyl and cyclohexyl.

The term “alkenyl” refers to a straight or branched hydrocarboncontaining one or more double bonds, preferably one or two double bonds.The term “C2-12 alkenyl”, for instance, refers to such a groupcontaining from two to twelve carbon atoms. Examples of alkenyl includeallyl, 1-methylvinyl, butenyl, iso-butenyl, 1,3-butadienyl,3-methyl-2-butenyl, 1,3-butadienyl, 1,4-pentadienyl, 1-pentenyl,1-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl and1,3,5-hexatrienyl.

The term “alkynyl” refers to a straight or branched hydrocarboncontaining one or more triple bonds, preferably one or two triple bonds.The term “C2-12 alkynyl”, for instance, refers to such a groupcontaining from two to twelve carbon atoms. Examples include 2-propynyland 2- or 3-butynyl.

The term “alkoxy” as used alone or in combination refers to a straightor branched chain alkyl group covalently bound via an oxygen linkage(—O—) and the terms “C1-6 alkoxy” and “lower alkoxy” refer to suchgroups containing from one to six carbon atoms, such as methoxy, ethoxy,propoxy, isopropoxy, butoxy, t-butoxy and the like.

The term “cycloalkenyl” refers to cyclic alkenyl groups having a singlecyclic ring or multiple condensed rings, and at least one point ofinternal unsaturation, preferably incorporating 4 to 11 carbon atoms.Examples of suitable cycloalkenyl groups include, for instance,cyclobut-2-enyl, cyclopent-3-enyl, cyclohex-4-enyl, cyclooct-3-enyl,indenyl and the like.

The term “acyl” refers to groups H—C(O)—, alkyl-C(O)—, cycloalkyl-C(O)—,aryl-C(O)—, heteroaryl-C(O)— and heterocyclyl-C(O)—, where alkyl,cycloalkyl, aryl, heteroaryl and heterocyclyl are described herein.

The term “aryl” refers to carbocyclic (non-heterocyclic) aromatic ringsor ring systems. The aromatic rings may be mono- or bi-cyclic ringsystems. The aromatic rings or ring systems are generally composed of 5to 10 carbon atoms. Examples of suitable aryl groups include but are notlimited to phenyl, biphenyl, naphthyl, tetrahydronaphthyl, and the like.

The term “alkylene” refers to a saturated, branched or straight chain orcyclic hydrocarbon radical having two monovalent radical centers derivedby the removal of two hydrogen atoms from the same or two differentcarbon atoms of a parent alkane. For example, an alkylene group can have1 to 20 carbon atoms, 1 to 10 carbon atoms, or 1 to 6 carbon atoms.Typical alkylene radicals include, but are not limited to, methylene(—CH₂—), 1,1-ethyl (—CH(CH₃)—), 1,2-ethyl (—CH₂CH₂—), 1,1-propyl(—CH(CH₂CH₃)—), 1,2-propyl (—CH₂CH(CH₃)—), 1,3-propyl (—CH₂CH₂CH₂—),1,4-butyl (—CH₂CH₂CH₂CH₂—), and the like.

The term “alkenylene” refers to an unsaturated, branched or straightchain or cyclic hydrocarbon radical having two monovalent radicalcenters derived by the removal of two hydrogen atoms from the same ortwo different carbon atoms of a parent alkene. For example, andalkenylene group can have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or1 to 6 carbon atoms. Typical alkenylene radicals include, but are notlimited to, 1,2-ethylene (—CH═CH—).

The term “alkynylene” refers to an unsaturated, branched or straightchain or cyclic hydrocarbon radical having two monovalent radicalcenters derived by the removal of two hydrogen atoms from the same ortwo different carbon atoms of a parent alkyne. For example, analkynylene group can have 1 to 20 carbon atoms, 1 to 10 carbon atoms, or1 to 6 carbon atoms. Typical alkynylene radicals include, but are notlimited to, acetylene (—C≡C—), propargyl (—CH₂C≡C—), and 4-pentynyl(—CH₂CH₂CH₂C≡CH—).

The term “arylene” refers to an aryl radical having two monovalentradical centres derived from the removal of two hydrogen atoms from thesame or two different carbon or heteroatoms of a parent aryl group.

The terms “halo” and “halogen” refers to fluoro, chloro, bromo and iodogroups.

The term “halo alkyl” group has one or more of the hydrogen atoms on analkyl group replaced with halogens.

The term “heteroaryl” refers to a monovalent aromatic carbocyclic group,preferably of from 2 to 10 carbon atoms and 1 to 4 heteroatoms selectedfrom oxygen, nitrogen and sulfur within the ring. Preferably theheteroatom is nitrogen. Such heteroaryl groups can have a single ring(e.g., pyridyl, pyrrolyl or furyl) or multiple condensed rings (e.g.,indolizinyl, benzothienyl, or benzofuranyl).

The term “heterocyclyl” refers to a monovalent saturated or unsaturatedgroup having a single ring or multiple condensed rings, preferably from1 to 8 carbon atoms and from 1 to 4 hetero atoms selected from nitrogen,sulfur, oxygen, selenium or phosphorous within the ring.

The term “optionally substituted” means that a group may include one ormore substituents. One or more hydrogen atoms on the group may bereplaced by substituent groups independently selected from halogens (forexample halo alkyl such as —CF₃ or —CF₂H), C₁₋₆alkyl, C₂₋₆alkenyl,C₂₋₆alkynyl, —(CH₂)_(v)C₃₋₇cycloalkyl, —(CH₂)_(v)C₄₋₇cycloalkenyl,—(CH₂)_(v)aryl, —(CH₂)_(v)heterocyclyl, —(CH₂)_(v)heteroaryl,—C₆H₄S(O)_(q)C₁₋₆alkyl, —C(Ph)₃, —CN, —OR, —O—(CH₂)₁₋₆—R,—O—(CH₂)₁₋₆—OR, —OC(O)R, —C(O)R, —C(O)OR, —OC(O)NR′R″, —NR′R″, —NO₂,—NRC(O)R′, —NRC(O)NR′R″, —NRC(S)NR′R″, —NRS(O)₂R′, —NRC(O)OR′,—C(NR)NR′R″, —C(═NOR′)R, —C(═NOH)NR′R″, —C(O)NR′R″, —C(═NCN)—NR′R″,—C(═NR)NR′R″, —C(═NR′)SR″, —NR′C(═NCN)SR″, —CONRSO₂R′, —C(S)NR′R″,—S(O)_(q)R, —SO₂NR′R″, —SO₂NRC(O)R′, —OS(O)₂R, —PO(OR)₂ and —NO₂; wherev is 0-6, q is 0-2 and each R, R′ and R″ is independently selected fromH, C₁₋₆alkyl, C₂₋₆alkenyl, C₂₋₆alkynyl, C₃₋₇-cycloalkyl,C₄₋₇cycloalkenyl, aryl, heterocyclyl, heteroaryl, C₁₋₆alkylaryl,C₁₋₆alkylheteroaryl, and C₁₋₆alkylheterocyclyl, wherein the alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl,heteroaryl, C₁₋₆alkylaryl, C₁₋₆alkylheteroaryl, orC₁₋₆alkylheterocyclyl, may be optionally substituted with one to six ofsame or different groups selected from halogen, hydroxy, lower alkyl,lower alkoxy, —CO₂H, CF₃, CN, phenyl, NH₂ and —NO₂; or when R′ and R″are attached to the same nitrogen atom, they may, together with the atomto which they are attached, form a 5 to 7 membered nitrogen containingheterocyclic ring.

In the lists of possible functional groups defined herein, where a givengroup contains two or more subgroups, for example the subgroups may belisted in the format [subgroup A][subgroup B] as in alkylaryl, or[subgroup A][subgroup B][subgroup C] as in aryloxyalkyl, the order ofthe subgroups as they are listed above is not intended to be limited tothe order in which they are presented. Thus, a group with two subgroupsdefined as [subgroup A][subgroup B], such as alkylaryl, is intended toalso be a reference to a group with two subgroups defined as [subgroupB][subgroup A], such as arylalkyl.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

The term “diastereomer” refers to a stereoisomer with two or morecenters of chirality and whose molecules are not mirror images of oneanother. Diastereomers have different physical properties, e.g., meltingpoints, boiling points, spectral properties, and reactivities. Mixturesof diastereomers may separate under high resolution analyticalprocedures such as electrophoresis and chromatography.

The term “enantiomers” refer to two stereoisomers of a compound whichare non-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L or R and Sare used to denote the absolute configuration of the molecule about itschiral center(s). The prefixes d and l or (+) and (−) are employed todesignate the sign of rotation of plane-polarized light by the compound,with (−) or l meaning that the compound is levorotatory. A compoundprefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

As used herein, amino acids and derivatives thereof may be abbreviatedto their respective three letter or single letter codes as appropriate.For example, cysteic acid may be referred to herein as Cya, Cyst orCyst-SO₃H, aspartic acid may be referred to as Asp.

The salts of the compounds of the invention are preferablypharmaceutically acceptable, but it will be appreciated thatnon-pharmaceutically acceptable salts also fall within the scope of thepresent invention, since these are useful as intermediates in thepreparation of pharmaceutically acceptable salts.

It will be appreciated that the compounds of the invention, and thesalts thereof, can be presented in the form of pharmaceuticallyacceptable derivatives. The term “pharmaceutically acceptablederivative” includes pharmaceutically acceptable esters, prodrugs,solvates and hydrates of the compounds of Formula (I), or salts thereof.Pharmaceutically acceptable derivatives may include any pharmaceuticallyacceptable hydrate or any other compound or prodrug which, uponadministration to a subject, is capable of providing (directly orindirectly) a compound of the invention, or an active metabolite orresidue thereof.

The pharmaceutically acceptable salts include acid addition salts, baseaddition salts, and the salts of quaternary amines and pyridiniums. Theacid addition salts are formed from a compound of the invention and apharmaceutically acceptable inorganic or organic acid including but notlimited to hydrochloric, hydrobromic, sulfuric, phosphoric,methanesulfonic, toluenesulphonic, benzenesulphonic, acetic, propionic,ascorbic, citric, malonic, fumaric, maleic, lactic, salicylic, sulfamic,tartaric or amino acids. The counter ion of quaternary amines andpyridiniums include chloride, bromide, iodide, sulfate, phosphate,methanesulfonate, citrate, acetate, malonate, fumarate, sulfamate, andtartrate. The base addition salts include but are not limited to saltssuch as sodium, potassium, calcium, lithium, magnesium, ammonium andalkylammonium. Also, basic nitrogen-containing groups may be quaternisedwith such agents as lower alkyl halides, such as methyl, ethyl, propyl,and butyl chlorides, bromides and iodides; dialkyl sulfates likedimethyl and diethyl sulfate; and others. The salts may be made in aknown manner, for example by treating the compound with an appropriateacid or base in the presence of a suitable solvent.

The compounds of the invention may be in crystalline form and/or assolvates (e.g. hydrates) and it is intended that both forms be withinthe scope of the present invention. The term “solvate” is a complex ofvariable stoichiometry formed by a solute (in this invention, a compoundof the invention) and a solvent. Such solvents should not interfere withthe biological activity of the solute. Solvents may be, by way ofexample, water, ethanol or acetic acid. Methods of solvation aregenerally known within the art.

The term “pro-drug” is used in its broadest sense and encompasses thosederivatives that are converted in vivo to the compounds of theinvention. Such derivatives would readily occur to those skilled in theart, and include, for example, compounds where a free hydroxy group isconverted into an ester derivative or a ring nitrogen atom is convertedto an N-oxide. Examples of ester derivatives include alkyl esters,phosphate esters and those formed from amino acids, preferably valine.Any compound that is a prodrug of a compound of the invention is withinthe scope and spirit of the invention.

The term “pharmaceutically acceptable ester” includes biologicallyacceptable esters of compound of the invention such as sulphonic,phosphonic and carboxylic acid derivatives.

Thus, in another aspect of the invention, there is provided a prodrug orpharmaceutically acceptable ester of a compound of the invention or ofsalt thereof.

It will be appreciated that the compounds of the invention have at leastone asymmetric centre, and therefore are capable of existing in morethan one stereoisomeric form. The invention extends to each of theseforms individually and to mixtures thereof, including racemates. Theisomers may be separated conventionally by chromatographic methods orusing a resolving agent. Alternatively the individual isomers may beprepared by asymmetric synthesis using chiral intermediates. Where thecompound has at least one carbon-carbon double bond, it may occur in Z-and E-forms with all isomeric forms of the compounds being included inthe present invention.

The invention also includes where possible a salt or pharmaceuticallyacceptable derivative such as a pharmaceutically acceptable ester,solvate and/or prodrug of the above mentioned embodiments of theinvention.

In another aspect of the invention, there is provided a pharmaceuticalcomposition that comprises a therapeutically effective amount of one ormore of the aforementioned compounds or pharmaceutically acceptablesalts thereof, including pharmaceutically acceptable derivativesthereof, and optionally a pharmaceutically acceptable carrier ordiluent.

In another aspect, the present invention provides pharmaceuticalcompositions for use in the treatment or prevention of influenza viralinfection, the composition comprising an effective amount of a compoundof the invention, or a pharmaceutically acceptable salt thereof,including a pharmaceutically acceptable derivative thereof, andoptionally a pharmaceutically acceptable carrier or diluent.

The term “composition” is intended to include the formulation of anactive ingredient with encapsulating material as carrier, to give acapsule in which the active ingredient (with or without other carrier)is surrounded by carriers.

The pharmaceutical compositions or formulations include those suitablefor oral, rectal, nasal, topical (including buccal and sub-lingual),vaginal or parenteral (including intramuscular, sub-cutaneous andintravenous) administration or in a form suitable for administration byinhalation or insufflation. In one embodiment, the compounds of theinvention are formulated for inhalation, oral administration, intranasaladministration, intraperitoneal administration, intravenousadministration or intramuscular administration.

General considerations in formulation and/or manufacture ofpharmaceutical compositions agents can be found, for example, inRemington's Pharmaceutical Sciences, Sixteenth Edition. E. W. Martin(Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science andPractice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins,2005).

In one embodiment, the compositions and formulations in accordance withthe invention may be administered to a person in need thereof by anysuitable inhalation, insufflation or intranasal delivery method.Suitable methods for inhalation, insufflation or intranasaladministration would be well-known to a person skilled in the art.

In one embodiment, the compositions or formulations of compounds of theinvention may be in a form suitable for administration by inhalation orinsufflation. For example, the compositions or formulations of compoundsof the invention may be prepared as a dry powder, solution, suspensionor aerosol for inhalation or insufflation via the mouth or nose.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis of infections as described herein.

In one embodiment, the compositions or formulation of compounds of theinvention may be administered by means of a dry powder inhaler. Drypowder inhalers of types readily disclosed in the art may be used, aswell as micronized shavings of isostatically compressed tablets or discsof drug with or without inert binders such as lactose may be used. Thismethod of administration provides particularly rapid delivery to thelung. When the compound of the invention is provided in the form of adry powder, it may be presented alone or in admixture with a suitablepharmaceutically acceptable diluent such as starch, starch derivativessuch as hydroxypropylmethyl cellulose or polyvinylpyrrolidine (PVP),sugar derivatives such as mannitol or lactose. The powder compositionmay be presented in unit dose form, for example in capsules orcartridges of e.g. gelatin or formed plastic or blister packs from whichthe powder may be administered by means of an inhalation device, or inmultidose form from, for example, a powder reservoir.

For inhalation of droplets, mists and aerosols various devices such asnebulizers or pressurized aerosol generators are readily available. Inaddition, such devices can be metered to provide uniformity of dosing.

All inhalers may be designed and fashioned to deliver liquid or solidparticles to the nasal mucosa as well as the upper and/or lower airways.

In another embodiment, the compositions or formulation of compounds ofthe invention may be administered by inhalation or insufflation by meansof a wet aerosol inhaler. For example, the compositions or formulationof compounds of the invention may be prepared for administration by wetaerosol inhalation in a pre-metered inhaler.

In a further embodiment, the compositions or formulations of compoundsof the invention are in a form suitable for administration intranasally.The intranasal compositions disclosed herein can be administered as aspray or drop. Accordingly, suitable commercial packages containing theintranasal formulation can be in any spray container known in the art.In one or more embodiments, the formulations in accordance with theinvention may be administered via a spray device or container. Spraydevices in accordance with the invention may be single unit dose systemsor multiple dose systems, for example comprising a bottle, a pump and/oran actuator. Such spray devices are available commercially. Suitablecommercial spray devices include those available from Nemera, Aptar,Bespak and Becton-Dickinson. Other suitable means for administering thecompositions or formulations intranasally in accordance with theinvention include via a dropper, a syringe, a squeeze bottle, and anyother means known in the art for applying liquids to the nasal mucosa inan accurate and repeatable fashion.

The compounds of the present invention can also be administered in awide variety of oral and parenteral dosage forms. It will be obvious tothose skilled in the art that the following dosage forms may comprise,as the active component, either a compound of the invention or apharmaceutically acceptable salt of a compound of the invention.

The compositions or formulations of compounds of the invention may be inthe form of a sterile injectable preparation, such as a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned herein.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, such as a solution in 1,3-butane-diol or prepared as alyophilized powder. Among the acceptable vehicles and solvents that maybe employed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile fixed oils may conventionally be employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid may likewise be used in the preparationof injectables. Furthermore, formulations suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions which may contain anti-oxidants, buffers, bacteriostats andsolutes which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents and thickening agents.

The compositions or formulations of compounds of the invention may alsobe administered orally, for example, tablets, troches, lozenges, aqueousor oil suspensions, dispersible powders or granules, emulsions, hard orsoft capsules, syrups or elixirs may be prepared. Compositions intendedfor oral use may be prepared according to any method known to the artfor the manufacture of pharmaceutical compositions and such compositionsmay contain one or more agents including sweetening agents, flavouringagents, colouring agents and preserving agents, in order to provide apalatable preparation.

The compounds of the invention, together with a conventional adjuvant,carrier, or diluent, may be placed into the form of pharmaceuticalcompositions and unit dosages thereof, and in such form may be employedas solids, such as tablets or filled capsules, or liquids such assolutions, suspensions, emulsions, elixirs, or capsules filled with thesame, all for oral use, in the form of suppositories for rectaladministration; or in the form of sterile injectable solutions forparenteral (including subcutaneous) use.

Such pharmaceutical compositions and unit dosage forms thereof maycomprise conventional ingredients in conventional proportions, with orwithout additional active compounds or principles, and such unit dosageforms may contain any suitable effective amount of the active ingredientcommensurate with the intended daily dosage range to be employed.Formulations containing ten (10) milligrams of active ingredient or,more broadly, 0.1 to one hundred (100) milligrams, per tablet, areaccordingly suitable representative unit dosage forms.

For preparing pharmaceutical compositions from the compounds of thepresent invention, pharmaceutically acceptable carriers can be eithersolid or liquid. Solid form preparations include powders, tablets,pills, capsules, cachets, suppositories, and dispensable granules. Asolid carrier can be one or more substances which may also act asdiluents, flavouring agents, solubilisers, lubricants, suspendingagents, binders, preservatives, tablet disintegrating agents, or anencapsulating material.

In powders, the carrier is a finely divided solid that is in a mixturewith the finely divided active component.

In tablets, the active component is mixed with the carrier having thenecessary binding capacity in suitable proportions and compacted in theshape and size desired.

The powders and tablets preferably contain from five or ten to aboutseventy percent of the active compound. Suitable carriers are magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose, a low melting wax, cocoa butter, and the like.The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as carrier providing acapsule in which the active component, with or without carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid formssuitable for oral administration.

For preparing suppositories, a low melting wax, such as an admixture offatty acid glycerides or cocoa butter, is first melted and the activecomponent is dispersed homogeneously therein, as by stirring. The moltenhomogenous mixture is then poured into convenient sized moulds, allowedto cool, and thereby to solidify.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

Liquid form preparations include solutions, suspensions, and emulsions,for example, water or water-propylene glycol solutions. For example,parenteral injection liquid preparations can be formulated as solutionsin aqueous polyethylene glycol solution.

Sterile liquid form compositions include sterile solutions, suspensions,emulsions, syrups and elixirs. The active ingredient can be dissolved orsuspended in a pharmaceutically acceptable carrier, such as sterilewater, sterile organic solvent or a mixture of both.

The compounds according to the present invention may thus be formulatedfor parenteral administration (e.g. by injection, for example bolusinjection or continuous infusion) and may be presented in unit dose formin ampoules, pre-filled syringes, small volume infusion or in multi-dosecontainers with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulation agents such as suspending,stabilising and/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilisation from solution, for constitution witha suitable vehicle, e.g. sterile, pyrogen-free water, before use.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavours,stabilising and thickening agents, as desired.

Aqueous suspensions suitable for oral use can be made by dispersing thefinely divided active component in water with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, or other well known suspending agents.

Also included are solid form preparations that are intended to beconverted, shortly before use, to liquid form preparations for oraladministration. Such liquid forms include solutions, suspensions, andemulsions. These preparations may contain, in addition to the activecomponent, colorants, flavours, stabilisers, buffers, artificial andnatural sweeteners, dispersants, thickeners, solubilising agents, andthe like.

For topical administration to the epidermis the compounds according tothe invention may be formulated as ointments, creams or lotions, or as atransdermal patch. Ointments and creams may, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions may be formulated with an aqueous or oilybase and will in general also contain one or more emulsifying agents,stabilising agents, dispersing agents, suspending agents, thickeningagents, or colouring agents.

Formulations suitable for topical administration in the mouth includelozenges comprising active agent in a flavoured base, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert base such as gelatin and glycerin or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier.

Solutions or suspensions are applied directly to the nasal cavity byconventional means, for example with a dropper, pipette or spray. Theformulations may be provided in single or multidose form. In the lattercase of a dropper or pipette, this may be achieved by the patientadministering an appropriate, predetermined volume of the solution orsuspension. In the case of a spray, this may be achieved for example bymeans of a metering atomising spray pump. To improve nasal delivery andretention the compounds according to the invention may be encapsulatedwith cyclodextrins, or formulated with other agents expected to enhancedelivery and retention in the nasal mucosa.

Administration to the respiratory tract may also be achieved by means ofan aerosol formulation in which the active ingredient is provided in apressurised pack with a suitable propellant such as a hydrofluoroalkane(HFA) or a chlorofluorocarbon (CFC) for example dichlorodifluoromethane,trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. The aerosol may conveniently also contain asurfactant such as lecithin. The dose of drug may be controlled byprovision of a metered valve.

Alternatively the active ingredients may be provided in the form of adry powder, for example a powder mix of the compound in a suitablepowder base such as lactose, starch, starch derivatives such ashydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).Conveniently the powder carrier will form a gel in the nasal cavity. Thepowder composition may be presented in unit dose form for example incapsules or cartridges of, e.g., gelatin, or blister packs from whichthe powder may be administered by means of an inhaler.

In formulations intended for administration to the respiratory tract,including intranasal formulations, the compound will generally have asmall particle size for example of the order of 5 to 10 microns or less.Such a particle size may be obtained by means known in the art, forexample by micronisation.

When desired, formulations adapted to give sustained release of theactive ingredient may be employed.

The pharmaceutical preparations are preferably in unit dosage forms. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, such as packeted tablets, capsules, and powders in vials orampoules. Also, the unit dosage form can be a capsule, tablet, cachet,or lozenge itself, or it can be the appropriate number of any of thesein packaged form.

The invention also includes the compounds in the absence of carrierwhere the compounds are in unit dosage form.

The amount of the compound of the invention to be administered may be inthe range from about 0.01 mg to 2000 mg per day, depending on theactivity of the compound and the disease to be treated.

The amount of the compound required for use in treatment will vary withthe route of administration, the nature of the condition being treatedand the age and condition of the animal (including human patients), andwill ultimately be at the discretion of the attendant veterinarian orphysician.

For example, a suitable dose will be in the range of from about 0.1 mgto 100 mg/kg of body weight per day, preferably in the range of 0.1 mgto 50 mg/kg/day.

In some embodiments, the dosage for oral administration would be in therange 1 mg/kg/day to 100 mg/kg/day. The dose for injection would be inthe range of 1 mg/kg/day to 100 mg/kg/day. The dose for inhalation wouldbe in the range of 0.01 mg/kg/day to 1 mg/kg/day. In some preferredembodiments, the dose would be in the range of 5 mg-50 mg/kg for oral orinjection administration two to three times a day for a period of 1 to15 days, preferably 3 to 12 days, more preferably 5 to 10 days. In otherpreferred embodiments, the dose would be in the range of 0.1-0.5 mg/kgfor inhalation one to five times a day a period of 1 to 15 days,preferably 3 to 12 days, more preferably for period of 5 to 10 days.Alternatively, in other preferred embodiment, the dose would be in therange of 0.2 mg/kg-0.4 mg/kg for inhalation as a single dailyadministration for the duration of treatment. It is understood that thedesired dose may be administered in a single dose or as divided doseadministered at appropriate intervals, for example as two, three, fouror more sub-doses per day.

The compounds of the invention are conveniently administered in unitdosage form, for example containing 0.1 to 10 mg of active ingredientper unit dosage form.

While it is possible that, for use in therapy, the compounds may beadministered as the raw chemical, it is preferable to present the activeingredient as a pharmaceutical formulation. The invention thus furtherprovides a pharmaceutical formulation including the compounds togetherwith one or more pharmaceutically acceptable carriers thereof and,optionally, other therapeutic and/or prophylactic ingredients. Thecarrier(s) must be ‘acceptable’ in the sense of being compatible withthe other ingredients of the formulation and not deleterious to therecipient thereof.

Pharmaceutical formulations include those suitable for oral, rectal,nasal, or parenteral (including intramuscular, intradermal,sub-cutaneous and intravenous) administration or in a form suitable foradministration to the gastrointestinal tract, or in a form suitable tothe respiratory tract (including the nasal passages) for example byinhalation or insufflation or for intradermal or sub-cutaneousimplantation or for transdermal patch. The formulations may, whereappropriate, be conveniently presented in discrete dosage units and maybe prepared by any of the methods well known in the art. All methodsinclude the step of bringing into association the active compound with acarrier, such as a liquid carrier or finely divided solid carrier orcombinations thereof, and then, if necessary, shaping the product intothe desired formulation.

Liquids or powders for intranasal administration, tablets or capsulesfor oral administration and liquids for intravenous administration arethe preferred compositions.

The pharmaceutical preparations of the compounds according to thepresent invention may be co-administered with one or more other activeagents in combination therapy. For example the pharmaceuticalpreparation of the active compound may be co-administered (for example,separately, concurrently or sequentially), with one or more otherantiviral agents, antibiotics, and anti-inflammatory drugs.

The term “therapeutically effective amount” refers to that amount whichis sufficient to effect treatment, as defined above, when administeredto a subject, such as a mammal, including a human in need of suchtreatment. The therapeutically effective amount will vary depending onthe subject and disease state being treated, the severity of theaffliction and the manner of administration, and may be determinedroutinely by one of ordinary skill in the art.

The term “treatment” as used herein covers any treatment of a disease orillness in an animal, preferably a mammal, more preferably a human, andincludes: (i) preventing the disease or condition from occurring in asubject, (ii) inhibiting the disease or condition, (iii) relieving thedisease or condition, or (iv) relieving the conditions/symptoms causedby the disease.

The terms “preventing” and “prophylaxis” as used herein refer toadministering a medicament beforehand to avert or forestall theappearance of one or more symptoms of a disease or disorder. The personof ordinary skill in the medical art recognizes that the term “prevent”is not an absolute term. In the medical art it is understood to refer tothe prophylactic administration of a drug to substantially diminish thelikelihood or seriousness of a condition, or symptom of the conditionand this is the sense intended in this disclosure. As used in a standardtext in the field, the Physician's Desk Reference, the terms “prevent”,“preventing” and “prevention” with regard to a disorder or disease,refer to averting the cause, effects, symptoms or progression of adisease or disorder prior to the disease or disorder fully manifestingitself.

The terms “administer”, “administering” or “administration” in referenceto a compound, composition or formulation of the invention meansintroducing the compound into the system of the animal in need oftreatment. When a compound of the invention is provided in combinationwith one or more other active agents, “administration” and its variantsare each understood to include concurrent and/or sequential introductionof the compound and the other active agents.

EXAMPLES

Aspects disclosed herein are further described by the followingnon-limiting examples.

General Methods and Notes

Unless otherwise noted, NMR spectra were recorded on Bruker Avance 300.Xwin-NMR version 3.5 on DPX 300A.

Unless otherwise noted, mass spectra were recorded on Mass SpectrometerWaters Micromass ZMD using an ESI (electrospray ionisation probe), usingWater Mass Lynx NT software.

Unless otherwise noted, flash column chromatography was performed onsilica gel 60 F245 (E. Merck).

Thin Layer Chromatography (TLC) was performed on silica gel pre-coatedplates (E. Merck).

Unless otherwise noted, high-performance liquid chromatography (HPLC)were performed on a Waters Alliance 2690 separation module, using aWaters dual wavelength 2487 UV detector and Waters Millennium 32software.

General Procedure for Regulation of Laboratory Animals

Ethics regulation of relevant experiments utilising laboratory animalswas conducted in accordance with the approval of the InstitutionalAnimal Care and Use Committee of the correspondingInstitutions/Universities.

General Procedure for Plaque Reduction Assay

MDCK cells were seeded into six-well tissue culture plates and grown toconfluence by standard methods. Influenza viruses were diluted in aminimal volume of phosphate-buffered saline (PBS) supplemented with 0.2%BSA to yield an estimated titre of 50 to 100 PFU per well. Afterabsorption onto the MDCK cells for 1 h at 37° C. in a 5% CO₂ atmosphere,the viral inocula were aspirated and replaced with viral growth medium(minimal Eagle's medium supplemented with BSA, trypsin, andinsulin-transferrin-selenium) containing agar or agarose at an amount(generally 1 to 2%) sufficient to cause the medium to gel at roomtemperature. The plates were incubated at 37° C. in a CO₂ atmosphereuntil plaques developed (generally 2 to 4 days). The plaques werevisualized with a suitable stain (e.g., 0.4% crystal violet in formalsaline) before they were counted. Antiviral potency (EC₅₀) wasdetermined as the concentration of the compound in the medium thatreduced the plaque numbers to 50% of the value for the untreatedcontrol.

Materials and General Procedure for CPE Assay

Test compounds were dissolved in sterile water and stored at −20° C.Compounds were subsequently diluted in assay media (MEM+trypsin) todesired concentrations.

The assay media comprised Gibco 1 MEM (500 mL) supplemented with 1 mMMEM non-essential amino acids, 50 U-penicillin 50 μg/mL streptomycin, 1mM sodium pyruvate. Trypsin (TPCK-treated) was added to the media to afinal concentration of 1 μg/ml, prior to the assay being conducted.

MDCK cells (ATCC) were grown in growth media-Gibco 1×MEM (500 mL)supplemented with 1 mM MEM non-essential amino acids, 50 U-penicillin-50μg/mL streptomycin, 1 mM sodium pyruvate, and 10% FBS.

Viral stock was diluted in PBS.

The assay was conducted using a CellTitre 96 Aqueous One Solution CellProliferation Assay (Promega).

Unless otherwise noted, the following general method was utilised forCPE assays:

-   -   i) MDCK cells were grown to confluency in 96 well trays in        growth media;    -   ii) Media was removed;    -   iii) 46 μL of diluted virus was added per well, including to        mock infected wells;    -   iv) Plates were incubated at 37° C. for 1.5 h. During this time,        test compounds were prepared.    -   v) After 1.5 h, the viral inoculum was removed.    -   vi) Compounds to be tested were prepared, by perform 1:3 serial        dilution of compounds on the abovementioned plates:        -   a) 100 μL of assay media was added into Rows 3 to 11;        -   b) 150 μL of compounds was added to Row 2;        -   c) Titration of 50 μL from Row 2 to 10, then 50 μL was            discarded from Row 10.        -   d) Row 11 served as positive and negative control.    -   vii) Cells were incubated at 37° C. for 48 h-96 h.    -   viii) 48 h-96 h post infection, the media was removed.    -   ix) Media was replaced with 100 μL of assay media without        trypsin.    -   x) 20 μL per well of detection solution was added.    -   xi) Plates were incubated at 37° C. for a further 1 h.    -   xii) Absorbance of the plates was measure of 490 nm.

Example 1: Pre-Treatment of Virus with Test Compounds Prior to Infectionof MDCK Cells

Representative compounds of Formula (I) were utilised to assessanti-viral activity prior to infection of MDCK cells. MD345 [(PK2TCA(Z₀)₂-[D-Cya-SO₃H)₇—OH], MD345 [(PK2 TCA(Z₀)₂-[Cya)₇-OH], MD348[(CHTCA(Z₀)₂-D-Asp-(D-Cya)-D-Asp-(D-Cya)-D-Asp-(D-Cya)-OH] and MD348[(CHTCA(Z₀)₂-D-Asp-(D-Cya-SO₃H)-D-Asp-(D-Cya-SO₃H)-D-Asp-(D-Cya-SO₃H)—OH]were tested as representative compounds of Formula (I). Activity againsttwo viral strains were tested, A/Sydney/250/99 (H1N1), and A/Sydney/5/97(H3N2). Zanamivir was utilised as a control.

Conditions are detailed in Table 1 and a summary provided in FIG. 1.

TABLE 1 Summary of conditions for pre treatment of virus prior toinfection of MDCK cells Condition 1 Virus in PBS pre-adsorbed on MDCKcells at 37° C. for 1 hr, virus suspension removed, then cells werewashed with PBS (x 1), added assay media, and incubated at 37° C. for 72hrs, tested by MTT colorimetric assay system. Condition 2 Virus weretreated with PBS at R.T. for 60 min, then preadsorbed on MDCK cells at37° C. for 1 hr. virus suspension removed. The cells were washed withPBS (x 1), assay media added, and incubated at 37° C. for 72 hrs, testedby MTT colorimetric assay system. Condition 3 Virus were treated withTrypsin (11 tg/m1) at R.T. for 60 min, then preadsorbed on MDCK cells at37° C. for 1 hr, virus suspension removed, afterwards cells were washedwith PBS (x 1), assay media added, and incubated at 37° C. for 72 hrs,tested by MTT colorimetric assay system. Condition 4 Virus were treatedwith Trypsin (1 μg/m1) at R.T. for 30 min, then mixed with compound atR.T. for 30 min, afterwards preadsobed on MDCK cells at 37° C. for 1 hr.virus suspension removed. The cells were washed with PBS (x, 1), assaymedia added, and incubated at 37° C. for 72 hrs, tested by MTTcolorimetric assay system. Condition 5 Virus were treated with compoundat R.T. for 30 min, then mixed with Trypsin (1 μg/m1) at RT. for 30 min,afterwards preadsorbed on MDCK cells at 37° C. for 1 hr. virussuspension removed. The cells were washed with PBS (x 1), assay mediaadded, and incubated at 37° C. for 72 hrs, tested by MTT colorimetricassay system. Condition 6 Virus, compound and Trypsin (1 μg/m1) weremixed at R.T. for 1 hr, then preadsorbed on MDCK cells at 37° C. for 1hr. virus suspension removed. The cells were washed with PBS (x 1),assay media added, and incubated at 37° C. for 72 hrs, tested by MTTcolorimetric assay system. Condition 7 Virus mixed with compound at R.T.for 1 hr, then preadsobed on MDCK cells at 37° C. for 1 hr, virussuspension removed. The cells were washed with PBS (x 1), assay mediaadded, and incubated at 37° C. for 72 hrs, tested by MTT colorimetricassay system.

Results for A/Sydney/250/99 are summarised in Table 2 and FIGS. 2A, 2Band 2C. Results for A/Sydney/5/97 are summarised in Table 3 and FIGS.3A, 3B and 3C.

The results indicate that compounds of Formula (I), MD345, and MD348,neutralized the viruses A/Sydney/25/99 (H1N1) and A/Sydney/5/97 (H3N2)extracellularly. Pretreatment of viruses with Zanamivir did not affectthe infectivity of viruses.

Subsequent time course comparison of MD345-2 and Zanamivir againstA/Sydney/250/99 indicates MD345-2 neutralized both virus in less than 1minute. Results for A/Sydney/250/99 are summarised in Table 2 and FIGS.2A, 2B and 2C. Results for A/Sydney/5/97 are summarised in Table 4 andFIG. 4.

TABLE 2 Pre treatment of virus prior to infection of MDCK cells Testcompounds: MD348-2, MD345-2 and Zanamivir (control) Virus Titre:A/Sydney/25/99 MD348-2 Conditions Average 1 0.368 0.404 0.332 0.368 20.386 0.307 0.372 0.355 3 0.361 0.347 0.326 0.345 4 1.963 2.507 2.442.303 5 2.501 2.386 2.444 6 2.301 2.26 2.29 2.284 7 2.083 2.407 2.4132.301 No infection 2.236 2.268 2.285 2.263 MD348-2 % over Control (noInfection) 1 16% 2 16% 3 15% 4 102% 5 108% 6 101% 7 102% No Infection100% MD345-2 Conditions Average 1 0.368 0.404 0.332 0.368 2 0.386 0.3070.372 0.355 3 0.361 0.347 0.326 0.345 4 2.384 2.468 2.219 2.357 5 2.372.109 2.277 2.252 6 2.305 2.303 2.206 2.271 7 2.203 2.14 2.168 2.170 Noinfection 2.236 2.268 2.285 2.263 MD345-2 % over Control (no Infection)1 16% 2 16% 3 15% 4 104% 5 100% 6 100% 7 96% No Infection 100% ZanamivirConditions Average 1 0.368 0.404 0.332 0.368 2 0.386 0.307 0.372 0.355 30.361 0.347 0.326 0.345 4 0.466 0.461 0.967 0.631 5 0.419 0.409 0.6250.484 6 0.446 0.512 0.443 0.467 7 0.663 0.791 0.727 No infection 2.2362.268 2.285 2.263 Zanamivir % over Control (no Infection) 1 16% 2 16% 315% 4 28% 5 21% 6 21% 7 32% No Infection 100% Notes: Compounds weredissolved in H₂O to 50 μg/mL. Further dilutions were made in 1 × PBS to5 μg/mL. Cells were maintained in media containing 1 μg/mL trypsin.Assayed at 72 h post infection. (1000 140711) 0.5 × 10⁻⁴ viruspre-adsorbed for 1 h, virus removed, cells washed once and fresh mediaadded, incubated for 72 h.

TABLE 3 Pre treatment of virus prior to infection of MDCK cells Testcompounds: MD348-2, MD345-2 and Zanamivir (control) Virus Titre:A/Sydney/5/97 MD348-2 Conditions Average 1 0.296 0.308 0.299 0.301 20.283 0.298 0.291 0.291 3 0.269 0.277 0.262 0.269 4 1.82 2.526 2.6822.343 5 2.6 2.488 2.625 2.571 6 2.69 2.48 2.658 2.609 7 2.597 2.46 2.692.582 No infection 2.581 2.501 2.581 2.554 MD348-2 % over Control (noInfection) 1 12% 2 11% 3 11% 4 92% 5 101% 6 102% 7 101% No Infection100% MD345-2 Conditions Average 1 0.296 0.308 0.299 0.301 2 0.283 0.2980.291 0.291 3 0.269 0.277 0.262 0.269 4 2.547 2.621 2.451 2.540 5 2.4442.473 2.486 2.468 6 2.568 2.523 2.456 2.516 7 2.491 2.437 2.437 2.455 Noinfection 2.581 2.501 2.581 2.554 MD345-2 % over Control (no Infection)1 12% 2 11% 3 11% 4 99% 5 97% 6 98% 7 96% No Infection 100% ZanamivirConditions Average 1 0.296 0.308 0.299 0.301 2 0.283 0.298 0.291 0.291 30.269 0.277 0.262 0.269 4 0.329 0.322 0.322 0.324 5 0.329 0.325 0.3030.319 6 0.32 0.336 0.324 0.327 7 0.322 0.298 0.293 0.304 No infection2.581 2.501 2.581 2.554 Zanamivir % over Control (no Infection) 1 12% 211% 3 11% 4 13% 5 12% 6 13% 7 12% No Infection 100% Notes: Compoundswere dissolved in H₂O to 50 μg/mL. Further dilutions were ade in 1 × PBSto 5 μg/mL. Cells were maintained in media containing 1 μg/mL trypsin.Assayed at 72 h post infection. (2000 271011) 1 × 10⁻⁴ viruspre-adsorbed for 1 h, virus removed, cells washed once and fresh mediaadded, incubated for 72 h.

TABLE 4 Time course comparison for pre treatment of virus prior toinfection of MDCX cells Test compounds: MD345-2 and Zanamivir (control)Virus Titre: A/Sydney/250/99 MD345-2 Time (min) Condition 3 AverageCondition 6 Average 1 0.335 0.354 0.395 0.361 1.752 1.664 1.759 1.725 50.337 0.366 0.367 0.357 1.856 1.723 1.727 1.769 10 0.354 0.363 0.3370.351 1.764 1.724 1.717 1.735 20 0.354 0.373 0.359 0.362 1.705 1.6451.626 1.659 25 0.311 0.437 0.346 0.365 1.588 1.539 1.693 1.607 30 0.3140.347 0.331 1.762 1.756 1.746 1.755 Control 1.829 1.71 1.625 1.721 1.8291.71 1.625 1.721 Zanamivir Time (min) Condition 3 Average Condition 6Average 1 0.356 0.318 0.337 0.338 0.798 0.568 5 0.347 0.355 0.351 0.3940.609 0.502 10 0.323 0.37 0.347 0.326 0.487 0.407 20 0.347 0.358 0.3530.461 0.728 0.595 25 0.411 0.339 0.375 0.375 0.322 0.349 30 0.383 0.3730.378 0.357 0.353 0.355 Control 1.721 1.721 MD345-2 Zanamivir Time (min)Condition 3 Condition 6 Condition 3 Condition 6 1 21% 100% 20% 33% 5 21%103% 20% 29% 10 20% 101% 20% 24% 20 21% 96% 20% 35% 25 21% 93% 22% 20%30 19% 102% 22% 21% Note: Compounds were dissolved in H₂O to 50 μg/mL.Further dilutions were made in 1 x PBS to 0.5 μg/mL. Cells weremaintained in media containing 1 μg/ml trypsin. Assayed at 72 h postinfection. (1000 140711) 1 × 10⁻⁴ virus pre-adsorbed for 1 h, virusremoved, cells washed once and fresh media added, incubated for 72 h.

Example 2: Comparison of Variable Acidic/Anionic Groups on AntiviralActivity of Test Compounds

Representative compounds of Formula (I) were utilised to compare theeffect of acidic/anionic groups on antiviral activity. The followingcompounds were tested as representative compounds of Formula (I):

-   -   MD314-1:        (CHTCA(Z₀)₂-L-Asp-(L-Cyst-SO₃H)-Asp-(L-Cyst-SO₃H)-L-Asp-(L-Cyst-SO₃H)—OH,        comprising three sulfonic acids and four carboxylic acids;    -   MD021-7: CHTGA(Z)₂-L-Asp, comprising containing 2 carboxylic        acids; and    -   MD051-3: PK2 TCA(Z)₂-L-Asp-L-Asp, comprising 3 carboxylic acids.

Activity against three viral strains were tested. A/Sydney/250/99(H1N1). A/Mississippi/03/01 (H1N1) Wild Type, and A/Mississippi/03/01(H1N1) H274Y (Oseltamivir resistant).

Results for A/Sydney/5/97 (H3N2) are summarised in Table 5 and FIG. 5.Results for A/Mississippi/03/01 (H1N1) Wild Type are summarised in Table5 and FIGS. 6 and 7. Results for A/Mississippi/03/01 (H1N1) H274Y(Oseltamivir resistant) are summarised in Table 5 and FIGS. 8 and 9.

TABLE 5 Comparison of acidic/anionic groups on antiviral activityA/Mississippi/03101 A/Mississippi/03/01 (H1N1) H274Y Test A/Sydney/5/97(H1N1) (Oseltamivir Compound (H3N2) Wild Type resistant) MD314-1 0.535ng/mL 0.18 ng/mL 0.18 ng/mL (0.27 nM) (0.09 nM) (0.09 nM) MD021-7 1.65ng/mL 0.535 ng/mL 0.535 ng/mL (1.32 nM) (0.43 nM) (0.43 nM) MD051-30.156 ng/mL 0.535 ng/mL 0.535 ng/mL (0.45 nM) (0.406 nM) (0.406 nM)

The results indicate that that the antiviral activities of the compoundswere proportional to the number of acidic/anionic groups.

Example 3: Modification of Viral Clearance Rates in the Lung and NasalTurbinates

The effect of representative compounds of Formula (I) on rates of viralclearance in the lung and nasal turbinates of mice was assessed. MD021(CHTCA(Z)₂-L-Asp) was tested as a representative compound of Formula(I). Zanamivir and PBS were utilised as controls.

20 mice per group were treated intranasally under anaesthesia with 2 μgMD021, 2 μg of Zanamivir or PBS at days −1, +1, +2, +3, +4 and +5 andinfected with a sub-lethal dose of 50 pfu of PR8 influenza virus on day0. Mice were weighed daily. Five mice per treatment group were killed ondays 1, 3, 5 and 7 post infection and their lungs and nasal turbinateswere harvested. Lungs were assayed for viral load. Nasal turbinates werestored at −80° C. until analysis.

All treatments, infection and killing of mice occurred within a two hourwindow 24 hours after the intervention of the day before. Mice did notreceive treatment on the same day they were killed. The timing oftreatments infection and organ harvest are summarised in FIG. 10.

Results are detailed in Table 6 and FIGS. 11 to 14. At all time points,lung viral load (expressed as log₁₀) in MD021 treated mice wassignificantly lower than that for PBS treated mice. At Days 3 and 7 postinfection lung viral load MD021 treated mice was also significantlylower than that for Zanamivir treated mice. Results were assessed usinga one-way ANOVA with Tukey post-test on day 1, 3 and 5 data and a MannWitney t-test on day 7 data. The threshold of the assay was 10^(1.07).Negative samples assigned value of 10¹. Untreated mice (PBS controltreatment) did not survive to day 7 despite the low dose of virus given.

Weight loss was assessed for groups of mice that were not killed untilDay 7 post-infection. Results indicate minimal weight loss aftertreatment with MD021, substantially less that weight loss observed withZanamivir administration at later time points.

TABLE 6 Modification of viral clearance rates in the lung and nasalturbinates MD021 Zanamivir PBS Individual titres Day 1 1.00 2.73 2.562.52 3.07 1.82 2.08 2.66 3.71 1.00 2.96 3.01 1.00 1.57 3.79 Day 3 1.824.44 6.12 2.33 4.66 5.68 2.51 4.97 5.90 4.31 4.19 6.12 2.63 3.16 5.94Day 5 2.85 4.84 5.75 3.77 3.00 5.84 3.59 4.97 5.99 3.82 5.77 6.00 4.305.66 5.34 Day 7 3.41 4.70 3.18 5.14 3.22 3.90 3.16 4.64 1.00 5.24 Groupmean titres Day 1 1.52 2.60 2.98 Day 3 2.72 4.28 5.95 Day 5 3.67 4.855.98 Day 7 2.79 4.72

Statistical analysis of the weight loss curves by 2-way repeatedmeasures ANOVA showed a highly significant difference in the treatmentcurves (p<0.0001). Note, 2-way repeated measures ANOVA was onlycompleted for data acquired up until day 5 post infection as the PBSgroup did not have complete data on days 6 and 7 due to premature death.Bonferroni post-test revealed that both Zanamivir and MD021 weresignificantly different from PBS on days 3, 4 and 5 (p<0.001). The sameanalysis performed on the complete weight loss curves for MD021 andZanamivir only revealed a statistically significant difference in thetwo weight loss curves overall (p=0.0443) with the Bonferroni post-testshowing that the significant differences occurred on day 6 (p<0.01) andday 7 (p<0.001).

The results indicate that multiple daily treatments with 2 μg ofcompound MD021 were able to reduce lung viral load significantlycompared to mice treated similarly with PBS at days 1, 3 and 5 postinfection and also with 2 μg of Zanamivir at day 3 and 7 post infection.The significant reduction in viral load was accompanied by less weightloss in the mice and is expected to equate to substantial reduction indisease severity. The minimal weight loss with MD021 throughout theentire disease course was striking and significantly better thanZanamivir at later time points.

Although mice were given a dose of PR8 virus that, in normalcircumstances, is sub-lethal, all mice receiving PBS control treatmentlost weight and were killed prior to day 7 post infection. Theunexpected premature deaths may be associated with detrimental effect ofdaily anaesthetic and intranasal administration of 50 μL of liquid.However, the MD021 and Zanamivir groups, treatment outweighed anydetrimental effects.

Example 4: CPE Assay Screen

CPE assay screens were conducted for representative compounds of Formula(I) in accordance with the general procedure for CPE assay outlinedabove. Results are detailed in Tables 7 and 8. Specific in vitroantiviral screens were also conducted for a range of viral strains,including Influenza A H7N9 A/Anhui/1/2003 (Table 9), Influenza A H5N1Hong Kong/213/2003 (Table 10), Influenza A H3N2 Perth/16/2009 (Table11), Influenza A H7N9 A/Anhui/1/2003 (Table 12), Influenza A H1N1California 07/2009 (Table 13), Influenza A H5N1 Duck/MN/1525/81 (Table14), Influenza A H5N1 Thailand/16/2004 (Table 15), Influenza A virusH1N1, A/Mississippi/3/2001 H275Y, Oseltamivir Resistant (Table 16),Influenza A virus H5N1 Duck/MN/1525/81 (Table 17). Influenza B virus.B/Brisbane/60/2008 (Table 18), Influenza B virus, B/Florida/4/2006(Table 19) and A/Sydney/250/99 (H1N1) (Table 20).

Anti-influenza activity data indicates that the more acidic groups incompounds of Formula (I) the higher the antiviral activity.

The anti-influenza activity of representative compounds of Formula (I)were further assessed in subsequent CPE assays (Table 38) and atdiffering viral titres (Table 39).

Example 5: Anti Influenza Virus Activity Against Oseltamivir ResistantStrains

The anti influenza virus activity against Oseltamivir resistant strainswas assessed for representative compounds of Formula (I). Inhibition ofplaque size and plaque number were recorded. Oseltamivir was utilised acontrol. Results are detailed in Table 21.

Example 6: Viral Plaque Reduction Assay

Representative compounds of Formula (I) were assessed in a viral plaquereduction against each of CA/07/2009 virus H1N1 and A/PR/8 virus H1N1.Zanamivir was utilised a control. Results are detailed in Table 22 and23, respectively.

Example 7: In Vivo Mouse Efficacy Data for Representative Compounds ofFormula (I)

The in vivo efficacy of representative compounds of Formula (I) wasassessed in mice against a range of viral infections.

Table 24 details mouse survival after virus infection with one ofA/California/04/2009 (H1N1) A/Victoria/3/75 (H3N2), A/Mississippi/3/01H275Y, Oseltamivir resistant virus (H1N1), A/Duck/MN/1525/81 (H5N1), orB/Sichuan/379/99 (FluB) and single compound treatment withrepresentative compounds of Formula (I) at different dosages.

Table 25 details mouse survival after intranasal virus infection byA/PR/8 virus (500 pfu/mouse) and single compound treatment withrepresentative compounds of Formula (I) at different dosages and times.

Table 26 details the effects of single intranasal treatment withrepresentative compounds of Formula (I) when administered at differentdosages post-infection on survival from an influenzaA/California/04/2009 (H1N1pdm) virus infection in mice. Zanamivir wasused as a control and saline was used as a placebo.

Table 27 details the effects of single intranasal treatment withrepresentative compounds of Formula (I) when administered at differentdosages 48 hrs post-infection on survival from an influenza A/PR/8(H1N1) virus infection in mice. Zanamivir was used as a control andsaline was used as a placebo.

Table 28 details the effects of single intranasal treatment withrepresentative compounds of Formula (I) when administered at 48 hrspost-infection on survival from an influenza A/PR/8 (H1N1) virusinfection in mice. Zanamivir was used as a control and saline was usedas a placebo.

Table 29 details the effects of single intranasal treatment withrepresentative compounds of Formula (I) when administered at differentdosages at 48 hrs post-infection on survival from an influenza A/PR/8(H1N1) virus (500 pfu/mouse) infection in mice. Zanamivir was used as acontrol and saline was used as a placebo.

Table 30 details the effects of single intranasal treatment withrepresentative compounds of Formula (I) when administered at differingdosages 60 hrs post-infection on survival from an influenza A/PR/8(H1N1) virus (500 pfu/mouse) infection in mice. Zanamivir was used as acontrol and saline was used as a placebo. Zanamivir was used as acontrol and saline was used as a placebo.

Table 31 details the effects of single intranasal treatment withrepresentative compounds of Formula (I) when administered at 60 hrspost-infection on survival from an influenza A/PR/8 (H1N1) virus (500pfu/mouse) infection in mice. Zanamivir was used as a control and salinewas used as a placebo.

Table 32 details the effects of single intranasal treatment withrepresentative compounds of Formula (I) when administered at 72 hrspost-infection on survival from an influenza A/PR/8 (H1N1) virus (500pfu/mouse) infection in mice.

Table 33 details the effects of single intranasal treatment withrepresentative compounds of Formula (I) when administered at 72 hrspost-infection on survival from an influenza A/PR/8 (H1N1) virus (500pfu/mouse) infection in mice.

Table 34 details the effects of single treatment with representativecompounds of Formula (I) when administered intraperitoneally at 1 hrbefore lethal challenge of influenza A/PR/8 (H1N1) virus (500 pfu/mouse)on mice.

Table 35 details the effects of treatment with representative compoundsof Formula (I) when administered intraperitoneally at 4 hrspost-infection (20 μg/mouse/day×5 days) on survival from an influenzaA/PR/8 (H1N1) virus (500 pfu/mouse) infection in mice.

The results presented in Tables 34 and 35 indicate that compounds ofFormula (I) can be used in systemic treatment of infection.

Table 36 details the effects of treatment with representative compoundsof Formula (I) when administered intranasally at 60 hrs post-infectionon survival from an influenza A/PR/8 (H1N1) virus (500 pfu/mouse)infection in mice.

Table 37 details the effects of treatment with representative compoundsof Formula (I) when administered intranasally at 60 hrs post-infectionon survival from an influenza A/PR/8 (H1N1) virus (500 pfu/mouse)infection in mice.

Table 38 details the effects of treatment with representative compoundsof Formula (I) when administered intraperitoneally at 1 hr before lethalchallenge of influenza A/PR/8 (H1N1) virus (500 pfu/mouse) on mice.

The results from in vitro and in vivo tests demonstrate that compoundsof Formula (I) are potent against a number of flu strains including fluA, B, avian flu and drug resistant strains. Furthermore, the resultsdemonstrate that compounds of Formula (I) did not exhibit cytotoxicity.

With regard to mouse studies, it was found that infected mice generallylost less body weight and/or recovered faster when treated withcompounds of Formula (I) when compared with either a saline placeboand/or a control (i.e. Zanamivir). Furthermore, it was found thatcompounds of Formula (I) were effective after administration to thenasal cave in mice for a period or more than 11 days.

The in vivo efficacy data indicates that the compounds of Formula (I)were at least 75 fold (at weight dosage) or >400 fold (at molar dosage)more active than a Zanamivir control.

With regard to in vivo efficacy data for mice infected with virus Flu APR8 (500 pfu/mouse) comparative time based intranasal administrations ofrepresentative compounds of the invention at 48 hrs, 60 hrs, or 72 hrsafter infection resulted in survival of the majority of mice, and amaximum body weight loss <15% (only three mice body weight loss between20-25%).

With regard to in vivo efficacy data for mice infected with virusInfluenza A/PR/8 virus (500 pfu/mouse), intraperitoneallyadministrations of representative compounds of the invention resulted insurvival of all mice, and indicates that compounds of the invention maybe used for systemic treatment of infection.

Two representative compounds of Formula (I), MD185 and MD317 were testedin a hERG IC₅₀ (hERG-CHO, automated patch-clamp) assay. Both resultsindicated that the two compounds were safe with regard to cardiactoxicity. Furthermore, MDI 85 and MD317 were tested in Kinome scan,there were no significant interaction between compounds and kinases at100 nM.

TABLE 7 CPE assay screen EC₅₀ EC₅₀ EC₅₀ EC₅₀ Compound CA/07/2009Duck/MN/1525/81 Perth/16/2009 Florida/4/2006 No. MW ng/ml nM ng/ml nMng/ml nM ng/ml nM MD015 1166 7 6 0.4 0.34 7.8 6.7 2.5 2.1 MD016 1166 97.7 0.6 0.51 3.3 2.8 8.2 7 MD021 1243 24 19.3 0.3 0.24 4.6 3.7 0.38 0.3MD051 1318 10 7.58 0.7 0.53 26 19.7 1.5 1.13 MD206 1187 2 1.68 0.7 0.597.3 6.1 >10000 >8.4 μM MD082 1110 7 6.3 0.4 0.36 8.4 7.5 15 13.5 MD1021274 10 7.85 >10000 >7.8 μM >10000 >7.8 μM >10000 >7.8 μM MDII21246 >2000 >1.6 μM 0.7 0.56 >10000   >8 μM 6.5 5.2 MD123 974 8 8.2 0.70.72 >10000  >10 μM 0.96 0.98 MD214 1340 7 5.22 0.2 0.15 >10000 >7.4 μM2.5 1.86 MD154 1485 9 6.06 <0.13 <0.087 <0.13 <0.087 <0.13 <0.087 MD2191712 5 2.92 0.5 0.29 >10000 >5.8 μM 1.7 0.99 MD242 1215 8 6.58 0.84 0.74.2 3.46 0.71 0.58 MD185 1852 6 3.2 <0.13 <0.07 <0.13 <0.07 <0.13 <0.07MD191 1457 9 6.1 <0.13 <0.089 <0.13 <0.089 0.32 0.21 Compound codeDescription of Structure Molecular Weight MD015 PK1 PYR(Z)₂ 1166 MD016PK2 PYR(Z)₂ 1166 MD021 CHTCA(Z)₂-L-Asp 1243 MD051 PK2TCA(Z)₂-L-Asp-L-Asp 1318 MD206 CHTCA(Z_(b))₂-L-Asp 1187 MD082 PK1Succinyl-SO₃H-(Z)₂ 1110 MD102 CHTCA(Z_(2b))₂(L-Asp)₂ 1274 MD112CHTCA(Z₃)₂(L-Asp)₂ 1246 MD123 C₈(Z₃)₂Suberic acid 974 MD214 PK1PYR(Z_(b))₂(D-Asp)₂ 1340 MD154 CHTCA(Z₀)₂-L-Asp(NHCH₂SO₃H)₂ 1485 MD219PK2 PYR(Z_(b))₂[(D-Asp)(NHCH₂SO₃H)₂]₂ 1712 MD242 CHTCA(Z_(p))₂-L-Asp1215 MD185 PK2 PYR(Z_(n))₂[D-Asp(NHCH₂SO₃H)₂]₂ 1852 MD191CHTCA(Z_(n))₂-D-Asp(NHCH₂SO₃H)₂ 1457 *CC₅₀ > 10,000 ng/mL MDCK cell line

TABLE 8 Anti-influenza virus activity derived from CPE assay* (EC₅₀ nM)MD185 MD314 MD345 MD348 MD021 Zanamivir Flu Virus (MW1852) (MW1982)(MW2201) (MW1982) (MW1243) (MW332) A/Sydney/250/99 0.29 0.077 0.0520.058 0.186 >198 A/Solomon Island/3/06 0.29 0.03 0.043 0.016 0.144 >44A/Townsville/74/2011 0.74 0.09 0.041 0.06 0.40 >15 A/Mississippi/03/01WT 0.89 0.09 0.24 0.27 0.43 >66 A/Mississippi/03/01H274Y 0.89 0.09 0.240.27 0.43 >66 A/Perth/261/2009 0.49 0.36 0.32 0.35 0.57 6.6A/Victoria/170/2012 2.7 1.0 0.53 0.83 2.0 >596 A/Sydney/5/97 2.7 0.410.83 1.26 2.0 >>24 A/Victoria/503/06 6.5 4.4 2.2 4.5 4.0 >198B/Townsville/2/2011 1.7 0.38 0.24 0.28 0.95 >1789 *CC₅₀ > 600 ng/ml MDCKcell line ** values are the means of at least three determinations

TABLE 9 In Vitro Antiviral Screen (Influenza A virus H7N9,A/Anhui/1/2003) EC₅₀ EC₉₀ CC₅₀ Compound No. Drug Assay Name (μg/ml)(μg/ml) (μg/ml) *SI₅₀ **SI₉₀ MD051 Virus yield reduction/Neutral Red(Toxicity) 0.0031 >1.0 >312 MD051 Neutral Red (Cytopathiceffect/Toxicity) <0.00032 >1.0 >3100 MD154 Virus yield reduction/NeutralRed (Toxicity) 0.01 >1.0 >100 MD154 Neutral Red (Cytopathiceffect/Toxicity) <0.00032 >1.0 >3100 MD185 Virus yield reduction/NeutralRed (Toxicity) 0.00185 >1.0 >540 MD185 Neutral Red (Cytopathiceffect/Toxicity) <0.00032 >1.0 >3100 Ribavirin Virus yieldreduction/Neutral Red (Toxicity) 8.25 >1000 >121 Ribavirin Neutral Red(Cytopathic effect/Toxicity) 24 >1000 >42 *SI₅₀ = CC₅₀/EC₅₀ **SI₉₀ =CC₅₀/EC₉₀

TABLE 10 In Vitro Antiviral Screen (Influenza A virus H5N1, HongKong/213/2003) EC₅₀ EC₉₀ CC₅₀ Compound No. Drug Assay Name (μg/ml)(μg/ml) (μg/ml) *SI₅₀ **SI₉₀ MD051 Virus yield reduction/Neutral Red(Toxicity) <0.00032 >1.0 >3100 MD051 Neutral Red (Cytopathiceffect/Toxicity) <0.00032 >1.0 >3100 MD154 Virus yield reduction/NeutralRed (Toxicity) <0.00032 >1.0 >3100 MD154 Neutral Red (Cytopathiceffect/Toxicity) <0.00032 >1.0 >3100 MD185 Virus yield reduction/NeutralRed (Toxicity) <0.00032 >1.0 >3100 MD185 Neutral Red (Cytopathiceffect/Toxicity) <0.00032 >1.0 >3100 Ribavirin Virus yieldreduction/Neutral Red (Toxicity) 14.68 >1000 >68 Ribavirin Neutral Red(Cytopathic effect/Toxicity) 17 >1000 >59 *SI₅₀ = CC₅₀/EC₅₀ **SI₉₀ =CC₅₀/EC₉₀

TABLE 11 In Vitro Antiviral Screen (Influenza A virus H3N2,Perth/16/2009) EC₅₀ EC₉₀ CC₅₀ Compound No. Drug Assay Name (μg/ml)(μg/ml) (μg/ml) *SI₅₀ **SI₉₀ MD185 Visual (Virus yield reductionlNeutralRed) 0.00086 >0.32 >370 MD185 Neutral Red (Cytopathic effect/Toxicity)0.0001 >0.32 >3200 MD317 Visual (Virus yield reduction/Neutral Red)0.00173 >0.32 >180 MD317 Neutral Red (Cytopathie effect/Toxicity)0.00032 >0.32 >1000 MD345 Visual (Virus yield reduction/Neutral Red)0.00485 >0.32 >66 MD345 Neutral Red (Cytopathic effect/Toxicity)0.00054 >0.32 >590 MD349 Visual (Virus yield reduction/Neutral Red)0.00115 >0.32 >280 MD349 Neutral Red (Cytopathic effect/Toxicity)0.0006 >0.32 >530 MD352 Visual (Virus yield reduction/Neutral Red)0.00208 >0.32 >150 MD352 Neutral Red (Cytopathic effect/Toxicity)0.00038 >0.32 >840 Ribavirin Visual (Virus yield reduction/Neutral Red)4.4 >320 >73 Ribavirin Neutral Red (Cytopathic effect/Toxicity)5 >320 >64 *SI₅₀ = CC₅₀/EC₅₀ **SI₉₀ = CC₅₀/EC₉₀

TABLE 12 In Vitro Antiviral Screen (Influenza A virus H7N9,A/Anhui/1/2003) EC₅₀ EC₉₀ CC₅₀ Compound No. Drug Assay Name (μg/ml)(μg/ml) (μg/ml) *SI₅₀ **SI₉₀ MD185 Visual (Virus yield reduction/NeutralRed) <0.0006 >2.0 >3333 MD185 Neutral Red (Cytopathic effect/Toxicity)0.001 >2.0 >2000 MD317 Visual (Virus yield reduction/Neutral Red)0.01 >2.0 >200 MD317 Neutral Red (Cytopathic effect/Toxicity)0.015 >2.0 >130 MD345 Visual (Virus yield reduction/Neutral Red)0.01 >2.0 >200 MD345 Neutral Red (Cytopathic effect/Toxicity)0.025 >2.0 >80 MD349 Visual (Virus yield reduction/Neutral Red)<0.0006 >2.0 >3333 MD349 Neutral Red (Cytopathic effect/Toxicity)0.0042 >2.0 >480 MD352 Visual (Virus yield reduction/Neutral Red)<0.0006 >2.0 >3333 MD352 Neutral Red (Cytopathic effect/Toxicity)0.00063 >2.0 >3200 Ribavirin Visual (Virus yield reduction/Neutral Red)2.20 >320 >267 Ribavirin Neutral Red (Cytopathic effect/Toxicity)6.4 >320 >50 *SI₅₀ = CC₅₀/EC₅₀ **SI₉₀ = CC₅₀/EC₉₀

TABLE 13 In Vitro Antiviral Screen (Influenza A virus H1N1,California/07/2009) EC₅₀ EC₉₀ CC₅₀ Compound No. Drug Assay Name (μg/ml)(μg/ml) (μg/ml) *SI₅₀ **SI₉₀ MD185 Visual (Virus yield reduction/NeutralRed) 0.00026 >0.32 >1230 MD185 Neutral Red (Cytopathic effect/Toxicity)0.00015 >0.32 >2100 MD317 Visual (Virus yield reduction/Neutral Red)0.0017 >0.32 >190 MD317 Neutral Red (Cytopathic effect/Toxicity)0.0009 >0.32 >360 MD345 Visual (Virus yield reduction/Neutral Red)0.0047 >0.32 >68 MD345 Neutral Red (Cytopathic effect/Toxicity)0.00077 >0.32 >420 MD349 Visual (Virus yield reduction/Neutral Red)0.0046 >0.32 >70 MD349 Neutral Red (Cytopathic effect/Toxicity)0.0007 >0.32 >460 MD352 Visual (Virus yield reduction/Neutral Red)0.0041 >0.32 >78 MD352 Neutral Red (Cytopathic effect/Toxicity)0.00025 >0.32 >1300 Ribavirin Visual (Virus yield reduction/Neutral Red)2.3 >320 >140 Ribavirin Neutral Red (Cytopathic effect/Toxicity)2.5 >320 >130 *SI₅₀ = CC₅₀/EC₅₀ **SI₉₀ = CC₅₀/EC₉₀

TABLE 14 In Vitro Antiviral Screen (Influenza A virus H5N1,Duck/MN/1525/81) EC₅₀ EC₉₀ CC₅₀ Compound No. Drug Assay Name (μg/ml)(μg/ml) (μg/ml) *SI₅₀ **SI₉₀ MD185 Visual (Virus yield reduction/NeutralRed) 0.00153 >0.32 >210 MD185 Neutral Red (Cytopathic effect/Toxicity)0.00033 >0.32 >970 MD317 Visual (Virus yield reduction/Neutral Red)0.00096 >0.32 >330 MD317 Neutral Red (Cytopathic effect/Toxicity)0.00099 >0.32 >320 MD345 Visual (Virus yield reduction/Neutral Red)0.0065 >0.32 >49 MD345 Neutral Red (Cytopathic effect/Toxicity)0.0018 >0.32 >180 MD349 Visual (Virus yield reduction/Neutral Red)0.00835 >0.32 >38 MD349 Neutral Red (Cytopathic effect/Toxicity)0.00085 >0.32 >380 MD352 Visual (Virus yield reduction/Neutral Red)0.0077 >0.32 >42 MD352 Neutral Red (Cytopathic effect/Toxicity)0.00094 >0.32 >340 Ribavirin Visual (Virus yield reduction/Neutral Red)2.3 >320 >140 Ribavirin Neutral Red (Cytopathic effect/Toxicity)6.5 >320 >49 *SI₅₀ = CC₅₀/EC₅₀ **SI₉₀ = CC₅₀/EC₉₀

TABLE 15 In Vitro Antiviral Screen (Influenza A virus H5N1,Thailand/16/2004) EC₅₀ EC₉₀ CC₅₀ Compound No. Drug Assay Name (μg/ml)(μg/ml) (μg/ml) *SI₅₀ **SI₉₀ MD185 Visual (Virus yield reduction/NeutralRed) 0.012 >2.0 >170 MD185 Neutral Red (Cytopathic effect/Toxicity)0.0045 >2.0 >440 MD317 Visual (Virus yield reduction/Neutral Red)0.010 >2.0 >200 MD317 Neutral Red (Cytopathic effect/Toxicity)0.0076 >2.0 >260 MD345 Visual (Virus yield reduction/Neutral Red)0.005 >2.0 >400 MD345 Neutral Red (Cytopathic effect/Toxicity)0.0069 >2.0 >290 MD349 Visual (Virus yield reduction/Neutral Red)0.010 >2.0 >200 MD349 Neutral Red (Cytopathic effect/Toxicity)0.0055 >2.0 >360 MD352 Visual (Virus yield reduction/Neutral Red)0.002 >2.0 >1000 MD352 Neutral Red (Cytopathic effect/Toxicity)0.0037 >2.0 >540 Ribavirin Visual (Virus yield reduction/Neutral Red)2.79 >320 >120 Ribavirin Neutral Red (Cytopathic effect/Toxicity)3.7 >320 >86 *SI₅₀ = CC₅₀/EC₅₀ **SI₉₀ = CC₅₀/EC₉₀

TABLE 16 In Vitro Antiviral Screen (Influenza A virus H1N1,A/Mississippi/3/2001 H275Y, Oseltamivir Resistant) EC₅₀ EC₉₀ CC₅₀Compound No. Drug Assay Name (μg/ml) (μg/ml) (μg/ml) *SI₅₀ **SI₉₀ MD185Visual (Virus yield reduction/Neutral Red) 0.006 >2.0 >330 MD185 NeutralRed (Cytopathic effect/Toxicity) 0.0062 >2.0 >320 MD317 Visual (Virusyield reduction/Neutral Red) <0.002 >2.0 >1000 MD317 Neutral Red(Cytopathic effect/Toxicity) <0.002 >2.0 >1000 MD345 Visual (Virus yieldreduction/Neutral Red) 0.0036 >2.0 >560 MD345 Neutral Red (Cytopathiceffect/Toxicity) 0.0036 >2.0 >560 MD349 Visual (Virus yieldreduction/Neutral Red) 0.0089 >2.0 >220 MD349 Neutral Red (Cytopathiceffect/Toxicity) 0.0067 >2.0 >300 MD352 Visual (Virus yieldreduction/Neutral Red) 0.008 >2.0 >250 MD352 Neutral Red (Cytopathiceffect/Toxicity) 0.0081 >2.0 >250 Ribavirin Visual (Virus yieldreduction/Neutral Red) 10 >320 >32 Ribavirin Neutral Red (Cytopathiceffect/Toxicity) 9.5 >320 >34 *SI₅₀ = CC₅₀/EC₅₀ **SI₉₀ = CC₅₀/EC₉₀

TABLE 17 In Vitro Antiviral Screen (Influenza A virus H5N1,Duck/MN/1525/81) EC₅₀ EC₉₀ CC₅₀ Compound No. Drug Assay Name (μg/ml)(μg/ml) (μg/ml) *SI₅₀ **SI₉₀ MD185 Visual (Virus yield reduction/NeutralRed) <0.002 >2.0 >1000 MD185 Neutral Red (Cytopathic effect/Toxicity)<0.002 >2.0 >1000 MD317 Visual (Virus yield reduction/Neutral Red)<0.002 >2.0 >1000 MD317 Neutral Red (Cytopathic effect/Toxicity)<0.002 >2.0 >1000 MD345 Visual (Virus yield reduction/Neutral Red)0.0042 >2.0 >480 MD345 Neutral Red (Cytopathic effect/Toxicity)0.0035 >2.0 >570 MD349 Visual (Virus yield reduction/Neutral Red)<0.002 >2.0 >1000 MD349 Neutral Red (Cytopathic effect/Toxicity)<0.002 >2.0 >1000 MD352 Visual (Virus yield reduction/Neutral Red)0.0023 >2.0 >870 MD352 Neutral Red (Cytopathic effect/Toxicity)0.0026 >2.0 >770 Ribavirin Visual (Virus yield reduction/Neutral Red)10 >320 >32 Ribavirin Neutral Red (Cytopathic effect/Toxicity)10 >320 >32 *SI₅₀ = CC₅₀/EC₅₀ **SI₉₀ = CC₅₀/EC₉₀

TABLE 18 In Vitro Antiviral Screen (Influenza B virus,B/Brisbane/60/2008) EC₅₀ EC₉₀ CC₅₀ Compound No. Drug Assay Name (μg/ml)(μg/ml) (μg/ml) *SI₅₀ **SI₉₀ MD185 Visual (Virus yield reduction/NeutralRed) 0.006 >2.0 >330 MD185 Neutral Red (Cytopathic effect/Toxicity)0.0062 >2.0 >320 MD317 Visual (Virus yield reduction/Neutral Red)<0.002 >2.0 >1000 MD317 Neutral Red (Cytopathic effect/Toxicity)<0.002 >2.0 >1000 MD345 Visual (Virus yield reduction/Neutral Red)0.0036 >2.0 >560 MD345 Neutral Red (Cytopathic effect/Toxicity)0.0036 >2.0 >560 MD349 Visual (Virus yield reduction/Neutral Red)0.0089 >2.0 >220 MD349 Neutral Red (Cytopathic effect/Toxicity)0.0067 >2.0 >300 MD352 Visual (Virus yield reduction/Neutral Red)0.008 >2.0 >250 MD352 Neutral Red (Cytopathic effect/Toxicity)0.0081 >2.0 >250 Ribavirin Visual (Virus yield reduction/Neutral Red)10 >320 >32 Ribavirin Neutral Red (Cytopathic effect/Toxicity)9.5 >320 >34 *SI₅₀ = CC₅₀/EC₅₀ **SI₉₀ = CC₅₀/EC₉₀

TABLE 19 In Vitro Antiviral Screen (Influenza B virus, B/Florida/4/2006)EC₅₀ EC₉₀ CC₅₀ Compound No. Drug Assay Name (μg/ml) (μg/ml) (μg/ml)*SI₅₀ **SI₉₀ MD185 Visual (Virus yield reduction/Neutral Red)0.006 >2.0 >330 MD185 Neutral Red (Cytopathic effect/Toxicity)0.0062 >2.0 >320 MD317 Visual (Virus yield reduction/Neutral Red)<0.002 >2.0 >1000 MD317 Neutral Red (Cytopathic effect/Toxicity)<0.002 >2.0 >1000 MD345 Visual (Virus yield reduction/Neutral Red)0.0036 >2.0 >560 MD345 Neutral Red (Cytopathic effect/Toxicity)0.0036 >2.0 >560 MD349 Visual (Virus yield reduction/Neutral Red)0.0089 >2.0 >220 MD349 Neutral Red (Cytopathic effect/Toxicity)0.0067 >2.0 >300 MD352 Visual (Virus yield reduction/Neutral Red)0.008 >2.0 >250 MD352 Neutral Red (Cytopathic effect/Toxicity)0.008 >2.0 >250 Ribavirin Visual (Virus yield reduction/Neutral Red)10 >320 >32 Ribavirin Neutral Red (Cytopathic effect/Toxicity)9.5 >320 >34 *SI₅₀ = CC₅₀/EC₅₀ **SI₉₀ = CC₅₀/EC₉₀

TABLE 20 In Vitro Antiviral Screen (A/Sydney/250/99) EC₅₀ Compound(ng/ml) EC₅₀ (nM) MD012 0.06 0.039 CHTCA(Z)₂(S₆) (MW 1534) MD021 0.030.024 CHTCA(Z)₂-L-Asp (MW 1243) Zanamivir (MW 332) 7.38 22.228

TABLE 21 Anti influenza virus activity against Oseltamivir resistantstrains Inhibition of plaque size and plaque number by representativecompounds of Formula (I) MD021 MD051 MD155 MD185 Oseltamivir ng/ml ng/mlng/ml ng/ml nM Virus Subtype Size Number Size Number Size Number SizeNumber Size Number A/Fukui H3N2 WT 0.1-1.0^(a) 1-10 0.1-10 0.1-1  0.1-1.0 0.1-10^(b ) 0.1-1.0 1 10-100 ≥1000 A/Fukui H3N2 E119V 0.10.1-1   0.1 0.1-1   0.1 0.1-1   0.1 0.1-1   100 ≥10,000 A/Perth pH1N1 WT1 10-100 0.1-1.0 1-10 0.1-1.0 1-10 1 1-10 10 100 A/Perth pH1N1 H275Y0.1-1.0 10-100 0.1-1.0 1-10 0.1-1.0 1-10 0.1-1.0   1-100^(b) 100-1000≥10,000 B/Perth B WT 1 1-10 1 1-10 0.1-1.0 1-10 1.0-10 1-10 100-10001,000 B/Perth B D197E 1  1-100 0.1-1.0  1-10^(b) 1.0-10  1-10 0.1-10 1-100 1000 ≥10,000 ^(a)A range is given when the IC₅₀ falls between twodrug concentrations. ^(b)Range seen with duplicates may span more thanone log₁₀ dilution.

TABLE 22 Viral Plaque Reduction Assay (CA/07/2009 virus H1N1) EC₅₀Compound MW ng/ml nM MD185 1852 0.095 0.05140 MD317 1318 0.068 0.05168MD343 1597 0.012 0.00749 MD344 2057 0.021 0.01038 MD345 2201 0.0170.00797 Zanamivir 332 1.969 5.933

TABLE 23 Viral Plaque Reduction Assay (A/PR/8 virus H1N1) EC₅₀ CompoundMW nM MD185 1852 4.256 MD344 2057 1.471 MD342 2208 1.276 MD345 22011.719 MD348 1982 1.919 MD051 1318 1.744 MD343 1597 1.273 MD317 13183.303 Zanamivir 332 10150

TABLE 24 In vivo mouse efficacy data for representative compounds ofFormula (I) Mice survivors/total after virus infection and compoundtreatment once at different dosages. MD021 MD154 MD185 Zanamivirμg/mouse μg/mouse μg/mouse μg/mouse Virus infected in mice 50 10 2 50 102 50 10 2 150 50 Placebo A/California/04/2009 (H1N1) 8/10 3/10 5/1010/10 10/10 4/10 10/10 9/10 4/10 2/10 0/20 A/Victoria/3/75 (H3N2) 10/10 5/10 6/10 8/10 3/10 0/20 A/Mississippi/3/01 H275Y 10/10  4/10 1/10 10/108/10 3/10 1/10 0/20 Oseltarnivir resistant virus (H1N1) 10/10  10/10 7/10 10/10 10/10 9/9  10/10 10/10  9/10 8/10 0/20 A/Duck/MN/1525/81(H5N1) 7/10 8/10 2/10 10/10  9/10 6/10 10/10 8/10 7/10 7/10 3/20B/Sichuan/379/99 (FluB) 8/10 8/10 7/10 10/10 10/10 9/10  9/10 9/10 8/109/10 1/20 Note: Compounds were administered once intranasally 24 hoursbefore infection; mice infected intranasally and observation period was21 days.

TABLE 25 In vivo mouse efficacy data for representative compounds ofFormula (I) Mice survivors/total after virus infection and compoundtreatment once at different dosages and times. Mice were infectedintranasally by A/PR/8 virus (500 pfu/mouse) and treated intranasallywith representative compounds of Formula (I). MD185 MD342 MD343 MD344MD345 MD348 Zanamivir placebo 0.25 μg/mouse 24 hrs before infection 4/55/5 4/5 4/5 5/5 4/5 0/5 0/5  0.5 μg/mouse 24 hrs before infection 5/55/5 5/5 5/5 5/5 5/5 0/5 0/5   2 μg/mouse 5 days before infection 5/5 5/55/5 5/5 5/5 0/5 0/5   5 μg/mouse 7 days before infection 5/5 5/5 5/5 5/50/5 0/5   5 μg/mouse 9 days before infection 4/5 5/5 5/5 5/5 5/5 5/5 0/50/5   5 μg/mouse 11 days before infection 4/5 5/5 4/5 2/5 5/5 5/5 0/50/5

TABLE 26 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intranasal treatment once with MD 185 andZanamivir when administered post-infection on survival from an influenzaA/California/04/2009 (H1N1pdm) virus infection in mice. Time oftreatment Mean Day of Compound μg/mouse (hours)^(a) Survivors/TotalDeath^(b) ± SD MD185 250 24 4/10   8.2 ± 1.2*** MD185 50 24 1/10   7.3 ±0.5*** MD185 10 24 0/10 6.3 ± 0.9 Zanamivir 250 24 0/9^(c ) 6.7 ± 0.7Placebo 24  1/13^(c) 5.6 ± 0.9 MD185 250 48   5/9^(c)** 6.5 ± 1.0 MD18550 48 3/10 7.7 ± 1.0 MD185 10 48 1/10  7.6 ± 0.9* Zanamivir 250 48 0/106.6 ± 0.8 Placebo 48 0/15 6.6 ± 0.6 ^(a)Relative to virus exposure.^(b)Of mice that died during the 21-day observation period. ^(c)Initialgroup sizes were 10 drug-treated and 15 placebo-treated mice. Fouranimals were excluded because they showed no evidence of infection (noweight loss). *P < 0.05, **P < 0.01, ***P < 0.001, compared to placebo.

TABLE 27 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intranasal treatment once with MD185, MD317,Zanamivir, or saline when administered at 48 hrs post-infection onsurvival from an influenzaA/PR/8 (H1N1) virus infection in mice. Time oftreatment Maximum mice Compound μg/mouse (hours)^(a) Survivors/Totalweight loss^(b) MD185 40 48 5/5 <10% 4/5; 12% 1/5 MD317 40 48 4/5 <10%2/5; 20% 2/5 Zanarnivir 40 48 0/5 Placebo Saline 48 0/5 MD185 100 48 5/5<10% 4/5; 17% 1/5 MD317 100 48 5/5 <10% 3/5;  15% 1/5; 20% 1/5 Zanamivir100 48 0/5 Placebo Saline 48 0/5 ^(a)Relative to virus exposure.^(b)During the 21 days observation period.

TABLE 28 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intranasal treatment once with MD185, MD345,MD348, Zanamivir, or saline when administered at 48 hrs post-infectionon survival from an influenzaA/PR/8 (H1N1) virus infection in mice. Timeof treatment Maximum mice Compound μg/mouse (hours)^(a) Survivors/Totalweight loss^(b) MD185 40 48 5/5 ≤5% 1/5; ≤10% 1/5; ≤15% 3/5 MD345 40 485/5 ≤10% 4/5;  ≤15% 1/5  MD348 40 48 5/5 ≤5% 1/5; ≤15% 3/5;  <25% 1/5Zanamivir 40 48  0/5^(c) Placebo Saline 48  0/5^(d) ^(a)Relative tovirus exposure; ^(b)During the 21 days observation period; ^(c)Mean dayof death: 6.9 days; ^(d)Mean day of death: 6.6 days

TABLE 29 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intranasal treatment once with MD185, MD348,Zanamivir, or saline when administered at 48 hrs post-infection onsurvival from an influenza A/PR/8 (H1N1) virus (500 pfu/mouse) infectionin mice. Time of treatment Maximum mice Compound μg/mouse (hours)^(a)Survivors/Total weight loss^(b) MD185 40 48 4/5   10% 1/5; <15% 1/5; 15%2/5 MD348 40 48 5/5 ≤10% 3/5;  15% 1/5; 25% 1/5 Zanamivir 40 48 0/5Placebo Saline 48 0/5 ^(a)Relative to virus exposure; ^(b)During the 21days observation period.

TABLE 30 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intranasal treatment once with MD185, Zanamivir,or saline when administered at 60 hrs post-infection on survival from aninfluenza A/PR/8 (H1N1) virus (500 pfu/mouse) infection in mice. Time oftreatment Maximum mice Compound μg/mouse (hours)^(a) Survivors/Totalweight loss^(b) MD185 40 60 5/5 ≤10% 3/5; 12% 1/5;   15% 1/5 MD185 20060 5/5  <10% 2/5; 12% 2/5;   15% 1/5 Zanamivir 200 60  4/5^(c)   10%1/5; 15% 1/5; ≥20% 2/5 Placebo Saline 60  0/5^(d) ^(a)Relative to virusexposure; ^(b)During the 21 days observation period; ^(c)Mean day ofdeath: 8 days; ^(d)Mean day of death: 6.6 days

TABLE 31 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intranasal treatment once with MD185, MD317,MD345, Zanamivir, or saline when administered at 60 hrs post-infectionon survival from an influenza A/PR/8 (H1N1) virus (500 pfu/mouse)infection in mice. Time of treatment Maximum mice Mean day of Compoundμg/mouse (hours)^(a) Survivors/Total weight loss^(b) death MD185 40 602/5 20% 1/5; 24% 1/5 7.3 days MD317 40 60 2/5 20% 1/5; 24% 1/5 7.0 daysMD345 40 60 5/5 18% 4/5; 20% 1/5 Zanamivir 2000 60 0/5 6.6 daysZanamivir 40 60 0/5 5.2 days Placebo Saline 60 0/5 5.2 days ^(a)Relativeto virus exposure; ^(b)During the 21 days observation period.

TABLE 32 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intranasal treatment once with MD185, Zanamivir,or saline when administered at 72 hrs post-infection on survival from aninfluenza A/PR/8 (H1N1) virus (500 pfu/mouse) infection in mice. Time oftreatment Maximum mice Mean day of Compound μg/mouse (hours)^(a)Survivors/Total weight loss^(b) death MD185 40 72 3/5  ≤5% 1/5; <15%1/5; <20% 1/5 7.25 days  MD185 200 72 4/5 <10% 1/5; <15% 2/5;  20% 1/57.5 days Zanamivir 40 72 0/5 5.8 days Zanamivir 200 72 0/5 6.2 daysPlacebo Saline 72 0/5   5 days ^(a)Relative to virus exposure;^(b)During the 21 days observation period.

TABLE 33 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intranasal treatment once with MD185, MD345,Zanamivir, or saline when administered at 72 hrs post-infection onsurvival from an influenza A/PR/8 (H1N1) virus (500 pfu/mouse) infectionin mice. Time of treatment Maximum mice Mean day of Compound μg/mouse(hours)^(a) Survivors/Total weight loss^(b) death MD185 40 72 6/10 20%2/10;  15% 1/10; 7.25 days  12% 2/10; 10/% 1/10  MD345 40 72 7/10 25%1/10;  18% 1/10; 7.0 days 15% 3/10; 12/% 2/10  Zanamivir 40 72 0/5  6.6days Placebo Saline 72 0/5  6.6 days ^(a)Relative to virus exposure;^(b)During the 21 days observation period.

TABLE 34 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intraperitoneal administration once with MD185,MD317, Zanamivir, or saline, 1 hr before lethal challenge of influenzaA/PR/8 (H1N1) virus (500 pfu/mouse) on mice. Time of treatment Maximummice Compound μg/mouse (hours)^(a) Survivors/Total weight loss^(b) MD18520 −1 5/5 15% 3/5; 18% 1/5; 20% 1/5 MD317 20 −1 4/5 15% 3/5; 24% 1/5 Zanamivir 20 −1  0/5^(c) Placebo Saline −1  0/5^(d) ^(a)Relative tovirus exposure; ^(b)During the 21 days observation period; ^(c)Mean dayof death: 7 days; ^(d)Mean day of death: 7 days

TABLE 35 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intraperitoneal administration 20 μg/mouse/day ×5 days with MD185, MD317, Zanamivir, or saline at 4 hrs post-infectionon survival from an influenza A/PR/8 (H1N1) virus (500pfu/mouse)infection in mice. Time of μg/mouse/ treatment Maximum mice Compound day× 5 (hours)^(a) Survivors/Total weight loss^(b) MD185 20 × 5 4 5/5  ≤10Zanamivir 20 × 5 4 0/5^(c) Placebo Saline × 5 4 0/5^(d) ^(a)Relative tovirus exposure; ^(b)During the 21 days observation period; ^(c)Mean dayof death: 7.5 days; ^(d)Mean day of death: 7.5 days.

TABLE 36 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intranasal treatment once with MD185, MD317,Zanamivir, or saline when administered at 60 hrs post-infection onsurvival from an influenza A/PR/8 (H1N1) virus (500 pfu/mouse) infectionin mice. Time of treatment Mean day of Compound μg/mouse (hours)Survivors/Total Maximum mice weight loss^(b) death MD185 40 60 5/5 10%4/5; 20% 1/5 MD317 40 60 5/5 10% 3/5; 24% 2/5 Zanamivir 2000 60 3/5 15%1/5  24% 2/5 8.5 days   Zanamivir 40 60 0/5 7 days Saline N/A 60 0/5 7days ^(a)Relative to virus exposure, ^(b)During the 21 days observationperiod

TABLE 37 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intranasal treatment once with MD345, MD356,MD357, MD358, MD359, Zanamivir, or saline when administered at 60 hrspost-infection on survival from an influenza A/PR/8 (H1N1) virus (500pfu/mouse) infection in mice Time of treatment Mean day of Compoundμg/mouse (hours)^(a) Survivors/Total Maximum mice weight loss^(b) deathMD345 40 60 1/5  5% 1/5 7 days MD356 40 60 5/5 <10% 1/5 <15% 3/5  25%1/5 MD357 40 60 4/5 <15% 3/5 20% 1/5 7 days MD358 40 60 5/5 <10% 2/5 15%3/5 MD359 40 60 4/5 <10% 1/5 17% 2/5 20% 1/5 7 days Zanamivir 40 60 0/56 days Saline N/A 60 0/5 6 days ^(a)Relative to virus exposure.^(b)During the 21 days observation period.

TABLE 38 In vivo mouse efficacy data for representative compounds ofFormula (I) Effects of intraperitoneal administration once with MD345,MD373, Zanamivir or saline, 1 hr before lethal challenge of influenzaA/PR/8 (H1N1) virus Time of Com- treatment Survivors/ pound (hours)^(a)Total Maximum mice weight loss^(b) MD345 −1 5/5 10% 1/5 12% 1/5 18% 1/522% 2/5 MD373 −1 5/5 10% 1/5 12% 1/5 20% 1/5 23% 2/5 Zanamivir −1 0/5^(c) Saline −1  0/5^(d) ^(a)Relative to virus exposure, ^(b)Duringthe 14 days observation period, ^(c)Mean day of death 7 days, ^(d)Meanday of death 5 days

TABLE 39 Anti-influenza virus activity in CPE assay for representativecompounds of Formula (I) (EC50 ng/ml/nM) Flu virus MD185 MD356 MD357MD358 MD359 MD371 MD021 A/Sydney/250/99 0.18/0.097  0.01/0.0054 0.02/0.0108 0.27/0.139 0.16/0.074  n/a 0.27/0.217 A/Soloman Island/3/060.06/0.032 0.03/0.016 0.04/0.021 0.09/0.046 0.05/0.023  n/a 0.06/0.048A/Townsville/74/2011 <0.03/<0.016 0.03/0.016 0.03/0.016  0.03/0.01540.01/0.0046 n/a 0.03/0.024 A/Mississippi/03/01/WT 0.08/0.043 0.06/0.0320.06/0.032 0.185/0.095  0.06/0.0278 0.06/0.035 0.06/0.048A/Mississippi/03/01/H274Y 0.18/0.097 0.06/0.032 0.133/0.086  0.03/0.0154 0.06/0.0278  0.02/0.0116 0.06/0.048 A/Perth/261/20090.09/0.048 n/a n/a 0.0065/0.0033  0.03/0.0139 0.009/0.0052 0.03/0.024A/Victoria/170/2012 0.27/0.145 0.27/0.145 1.20/0.647 n/a n/a 0.27/0.1570.27/0.217 A/Sydney/5/97 0.13/0.070 0.06/0.032 0.18/0.097 0.09/0.0460.16/0.074  n/a 0.27/0.217 B/Townsville/2/2011 5.07/2.73  0.28/0.151 2.5/1.348 2.5/1.28 2.5/1.158 n/a 2.5/2.01

TABLE 40 Anti-influenza virus activity in CPE assay for representativecompounds of Formula (I) at diflerent viral titres (EC₅₀ ng/ml/nM) Fluvirus MD185 MD356 MD358 MD359 MD371 MD345 MD021 A/Townsville/74/2011<0.03/<0.016 0.03/0.016 0.03/0.0154 0.01/0.0046 n/a 0.03/0.01360.03/0.24 (1 × 10⁻⁵/ml) A/Townsville/74/2011  66/35.6 0.8/0.43 48/24.7 66/30.5  66/38.6 158/71.7   No inhibition (5 × 10⁻⁵/ml) at 197

Example 8: ADME Toxicology for Representative Compounds of Formula (I)

Two compounds of Formula (I), MD185 and MD317, were tested in an hERGIC₅₀ (hERG-CHO, automated patch-clamp) assay. The IC₅₀ value of MD185was not calculable. Concentration-response curve for MD317 showed lessthan 25% effect at the highest validated testing concentration, i.e.IC₅₀>100 nM.

Results indicated that MD185 and MD317 were safe on cardiac toxicity.

Example 9: KINOME Scan Profiling for Representative Compounds of Formula(I)

Two compounds of Formula (I), MD185 and MD317, were tested according tothe following protocol for kinase assays.

For most assays, kinase-tagged T7 phage strains were grown in parallelin 24-well blocks in an E. coli host derived from the BL21 strain. E.coli were grown to log-phase and infected with T7 phase from a frozenstock (multiplicity of infection=0.4) and incubated with shaking at 32°C. until lysis (90-150 minutes). The lysates were centrifuged (6,000×g)and filtered (0.2 μm) to remove cell debris. The remaining kinases wereproduced in HEK-293 cells and subsequently tagged with DNA for qPCRdetection. Streptavidin-coated magnetic beads were treated withbiotinylated small molecule ligands for 30 minutes at room temperatureto generate affinity resins for kinase assays. The liganded beads wereblocked with excess biotin and washed with blocking buffer(SeaBlock(pierce), 1% BSA, 0.05% Tween20, 1 mMDTT) to remove unboundligand and to reduce non-specific phage binding. Binding reactions wereassembled by combining kinases, liganded affinity beads, and testcompounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween20, 6mMDTT). Test compounds were prepared as 40× stocks in 100% DMSO anddirectly diluted into the assay. All reactions were performed inpolypropylene 384-well plates in a final volume of 0.04 ml. The assayplates were incubated at room temperature with shaking for 1 hour andthe affinity beads were washed with buffer (1×PBS, 0.05% Tween 20). Thebeads were then resuspended in elution buffer (1×PBS, 0.05% Tween 20,0.5 μM non-biotinylated affinity ligand) and incubated at roomtemperature with shaking for 30 minutes. The kinase concentration in theeluates was measured by qPCR

There were no significant interaction between compounds and kinases at100 nM. Results are summarized in Table 41.

TABLE 41 Selectivity score table Selectivity Number of ScreeningCompound Score Number Non-Mutant Conc. Selectivity Name Type of HitsKinases (nM) Score MD185 S(35) 0 395 100 0 MD185 S(10) 0 395 100 0 MD185S(1)  0 395 100 0 MD317 S(35) 0 395 100 0 MD317 S(10) 0 395 100 0 MD317S(1)  0 395 100 0Preparation of Representative Compounds of the Invention

For convenience, many chemical moieties are represented using well knownabbreviations, including but not limited to, methyl (Me), ethyl (Et),n-propyl (nPr), isopropyl (iPr), n-butyl (nBu), tert-butyl (tBu),n-hexyl (nHex), cyclohexyl (cHex), phenyl (Ph), methoxy (MeO), ethoxy(EtO), trimethylsilyl (TMS), tert-butyloxycarbonyl (Boc), and acetyl(Ac).

For convenience, many chemical compounds are represented using wellknown abbreviations, including but not limited to, methanol (MeOH),ethanol (EtOH), diethyl ether (Et2O), ethyl acetate (EtOAc),triethylamine (TEA), dichloromethane (methylene chloride, DCM, CH₂Cl₂),trifluoroacetic acid (TFA), trifluoroethanol (TFE), dimethylformamide(DMF), sodium sulphate (Na₂SO₄), tetrahydrofuran (THF),meta-chloroperoxybenzoic acid (mCPBA), hexamethyldisilazane sodium salt(NaHMDS), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU),O-(benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate (HBTU), dimethylsulfoxide (DMSO), magnesium sulphate(MgSO₄), sodium hydrogen carbonate (NaHCO₃), tert-butanol (t-BuOH),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride salt(EDCl.HCl), tetra-n-butylammonium fluoride (TBAF),N,N-diisopropylethylamine (DIPEA), tert-butyldimethylsilyl (TBDMS),1-hydroxybenzotriazole (HOBt),trans-dichlorobis(triphenylphosphine)palladium(II) (PdCl₂(PPh₃)₂),tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄)tris(dibenzylideneacetone) dipalladium(0) (Pd₂(dba)₃), tri-t-butylphosphonium tetrafluoroborate (t-Bu₃PH.BF₄),4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos),triphenylphosphine (PPh₃), diisopropyl azodicarboxylate (DIAD),pyridinium chlorochromate (PCC), borane dimethylsulfide (BMS), titaniumisopropoxide (TiOiPr₄), sodium triacetoxyborohydride (NaBH(OAc)₃),sodium cyanoborohydride (NaBH₃(CN)), sodium borohydride (NaBH₄),ammonium chloride (NH₄Cl), chloroform (CHCl₃), manganese dioxide (MnO₂),potassium carbonate (K₂CO₃), 1,2-dichloroethane (DCE), sodium azide(NaN₃), sodium nitrite (NaNO₂) di-tert-butyl dicarbonate (Boc₂O), andS-Acetamidomethyl (Acm).

General synthesis of compounds of Formula (I) is summarised in Scheme 1below:

Example 10: Preparation of N-Boc-1,9-diaminononane

1,9-Diaminononane (1 g, 6.329 mmoles) was dissolved in a mixture of 50ml ethanol and 50 ml water, then was added di-t-butyldicarbonate (1.38g, 6.329 mmoles) at R.T. The whole mixture was stirred at R.T. for 16hrs to afford a white suspension. This suspension was filtered off. Thesolid was di-Boc-1,9-diaminononane 488 mg (1.36 mmoles, 21.53% yd.)after air dried. The filtrate was extracted with dichloromethane (150ml, 100 ml). The aqueous layer containing unreacted 1,9-diaminononane(289 mg, 1.83 mmoles) can be recycled into next batch of preparation.The organic extracts were combined and washed with 50 ml of water, thenstirred with 10 g anhydrous Na₂SO₄ at R.T. overnight. The organicsuspension was filtered. The filtrate was vacuum evaporated into drynessto afford N-Boc-1,9-diaminononane 809 mg (49.5% yd.) as a colorlesssolid. MS 295 (M+1).

Example 11: Preparation of Anchoring Compound Zn′

Synthesis is detailed in Scheme 2 below. The following compoundreference numbers and synthetic step references relate to Scheme 2.

Step A) Sialic acid (1) (5 g, 16.18 mmoles) and Dowex 50×8(H⁺) resin (10g) were stirred in anhydrous methanol (400 mL) at 60-62° C. for 48 hrs.The mixture was filtered off. The filtrate was vacuum evaporated todryness to afford Compound (2) as a white solid 4.5 g (13.25 mmoles,82.5% yd.). MS 338 (M+1).

Step B) Compound (2) (4 g, 11.87 mmoles) was stirred with aceticanhydride (40 mL, d 1.08, MW 102.29, 423 mmoles) and sulphuric acid (2mL). The mixture was then stirred at 32-35° C. for 72 hr in an oil-bath.The reaction mixture was added dropwise to a stirring mixture of Na₂CO₃(51 g) in water (280 mL) and ethyl acetate (14 mL) at an ice-bath.Afterwards, the mixture was stirred for additional 1.5 hr in anice-bath, an subsequently extracted with ethyl acetate (400 mL, 270 mL).The ethyl acetate extracts were combined and washed with 10% NaHCO₃solution (270 mL×2), saturated NaCl solution (270 mL×2), dried overanhydrous Na₂SO₄ overnight. Filtered off, the filtrate was vacuumevaporated to dryness to afford compound (3) 4.78 g (11.57 mmoles, 97.5%yd.). MS 414 (M+1). The oily residue turned into an off white solidafter storage in a desiccator over P₂O₅ for 3 days.

Step C) Compound (3) (3.47 g, 8.4 mmoles) was dissolved in tert-BuOH (25mL). To this solution was added azidotrimethylsilane (1.81 mL, 13.63mmoles). The whole mixture was stirred at 80-82° C. under argon for 24hrs. The reaction mixture was diluted with ethyl acetate (100 mL), thenstirred with 0.9 g NaNO₂ in 25 mL of water, and adjusted to pH 2 with 5NHCl over a period of 1 hr at room temperature. The two phase mixture wasseparated, the aqueous layer was extracted with ethyl acetate (50 mL).The organic extracts were combined and washed successively with water(25 mL×2), 6% NaHCO₃ solution (25 mL), water (25 mL×3), then dried overNa₂SO₄. The filtrate was vacuum evaporated to dryness to afford Compound(4) 3.17 g (6.95 mmoles, 82.7% yd.) as an oily substance. MS 457 (M+1),479 (M+Na), 925 (2M+Na).

Step D) Compound (4) (3.15 g, 6.91 mmoles) was dissolved in methanol(100 mL) and toluene (70 mL). The solution was placed under vacuum toremove air (oxygen), then backfilled with argon. To this mixture wasadded Pd/C (10%) (616 mg), then placed under vacuum to evacuate argonwhich was subsequently replaced with hydrogen (H₂). The hydrogenationwas carried out at room temperature for 2 hr and the catalyst wassubsequently filtered off. The filtrate was vacuum evaporated to drynessto afford compound (5) 2.82 g (6.55 mmoles, 94.7% yd.) as an off whitesolid. MS 431 (M+1).

Step E) Compound (5) (2.80 g, 6.51 mmoles) was dissolved in anhydrousacetonitrile (15 mL). To this solution was addedN,N′-Di-Boc-1H-pyrazole-1-carboxamidine [Bis(Boc)PCH] (3.03 g, 9.76mmoles). The whole mixture was stirred under argon at room temperaturefor 40 hr. The reaction mixture was vacuum evaporated to dryness. Theresidue was dissolved in ethyl acetate (4 mL), then diluted with hexane(4 mL), applied on flash column chromatography, washed with hexane (150mL), then with solvent (ethyl acetate:hexane 1:1) (150 mL), finallyeluted with solvent (ethyl acetate:hexane 1:1) Vacuum evaporation of thecollected fractions gave the product Compound (6), 3.23 g (4.8 mmoles,73.7% yd.) as a white solid. MS 673 (M+1).

Step F) Compound (6) (2.8715 g, 4.273 mmoles) was dissolved in anhydrousmethanol (22 mL). To this solution was added NaOCH₃ (4.9134 mg, 0.2136mmoles) under argon. The whole mixture was stirred under argon at R.T.for 2.5 hr, then adjusted to pH 6.5 with Dowex50×8(H⁺) resin, which wassubsequently filtered off. The filtrate was vacuum evaporated to drynessto give compound (7) 2.2024 g (4.0337 mmoles, 94.4% yd.) as a whitesolid. MS 547 (M+1).

Step G) Compound (7) (2 g, 3.66 mmoles) was dissolved in anhydrousacetonitrile. To this solution was added 1,1′-carbonyldiimidazole (714mg, 4.403 mmoles). The whole mixture was stirred under argon at 20-30°C. for 18 hr. It was vacuum evaporated to dryness, then the residue wassubjected to flash column chromatography, firstly washed with hexane(150 mL), then developed with solvent (ethyl acetate:hexane 2:1). Thefractions at R_(f) value of 0.5 (ethyl acetate:hexane 2:1) were combinedand vacuum evaporated to dryness to afford Compound (8) 1.35 g (2.36mmoles, 64.5% yd.) as a white solid. MS 573 (M+1).

Step H) Compound (8) (1.3 g, 2.27 mmoles) was dissolved in anhydrouspyridine (12.5 mL). To this solution were added p-nitrophenylchloroformate (503.3 mg, 2.497 mmoles), 4-dimethylamino-pyridine (808.5mg, 6.62 mmoles). The whole mixture was stirred at 30° C. under argonfor 7 hr. To this reaction mixture was added a solution ofN-Boc-1,9-diaminononane (690 mg, 2.67 mmoles) and 4-dimethylaminopyridine (166 mg, 1.36 mmoles) in anhydrous pyridine (4 mL). Thereaction mixture was stirred under argon at 30° C. for 16 hr, thenvacuum evaporated to remove pyridine. The residue was partitionedbetween ethyl acetate (300 mL) and water (50 mL) containing 2.45 mL of 5NHCl, washed successively with water (50 mL×2), 2% NaHCO₃ solution (50mL×6), water (50 mL×2), dried over anhydrous Na₂SO₄, and filtered. Thefiltrate was vacuum evaporated to dryness to give 2.25 g of an oilysubstance. It was subjected to flash column chromatography (ethylacetate:hexane 1.5:1 as eluent). The fractions (at a Rf value of 0.27TLC, ethyl acetate:hexane 1.5:1 as developing solvent) were combined andvacuum evaporated to dryness to afford compound (9) 1.057 g (1.234mmole, 54.4% yd.) as a white solid. MS 857 (M+1), 879 (M+Na). ¹H-NMR(DMSO-d₆) δ(ppm) 11.42 (1H, s, guanidine NHBoc), 8.25 (1H, d, NH-4),7.95 (1H, H-3), 4.92 (2H, m, H-7, H-8), 4.75 (1H, m, H-4), 4.31-4.62(4H, m, H-5, H-6, H-9, H-9), 3.74 (3H, s, COOCH₃), 2.90 (4H, m,NHCH₂(CH₂)₇CH₂NH), 1.86 (3H, s, NHCOCH₃), 1.49 (9H, s, Boc), 1.38 (18H,s, Boc), 1.2-1.6 (14H, m, NHCH₂(CH₂)₇CH₂NH).

Step I) Compound (9) (1 g, 1.168 mmoles) was dissolved in a mixture oftrifluoro acetic acid (TFA) (36 ml) and methyl phenyl ether (Anisole)(3.9 ml) in dichloromethane (CH₂Cl₂) (36 ml). The whole mixture wasstirred at 25° C. for 2 hr and 40 min, then it was vacuum evaporated at35° C. for 2 hrs. The residue was stirred in hexane (100 ml) at R.T.overnight, the hexane was decanted and fresh hexane (60 ml) was added,and stirring was continued for 4 hrs at R.T. The hexane was thenremoved. The residue was dissolved in CH₂Cl₂ (10 ml) and evaporated todryness at 35-40° C. The residue was dissolved in water (25 ml). Theaqueous solution was freeze dried to afford compound (10) 1.026 g (1.143mmoles, 97.8% yd.) as a white foam of TFA₃Z_(n′) salt. MS 557 (M+1) [MWof Z_(n′)=556, TFA₃Z_(n′)=898].

Example 12: Preparation of Anchor-Backbone Compounds PYR(Z_(n′))₂

Synthesis is detailed in Scheme 3 below. The following compoundreference numbers relates to Scheme 3.

To a solution of pyromellitic dianhydride (11) (27.25 mg, 0.125 mmoles)in anhydrous DMF (2 ml) were added Z_(n′) (10) (224.5 mg, 0.25 mmoles)and diisopropylethylamine (DIPEA) (130.64 μl, 0.75 mmoles) at roomtemperature. The whole reaction mixture was allowed to stir under argonat 25° C. for 36 hrs, then treated with ether:petroleum ether 1:1 (100ml). The precipitate was filtered, washed with ether, and air dried toafford an off-white solid (197 mg), which was then subjected to HPLCseparation and purification.

HPLC Analytical: Pepl Gradient

Column: Gemini C18 column 100A 5 μm 150×3.00 mm. Wavelength: 220/280 nm.Flow rate: 0.7 ml/min. Solvent A=0.1% Trifluoroacetic acid. SolventB=100% Acetonitrile. Temperature: 30° C. Gradient: 0-50% B, 15 minutes.Retention time: PK1PYR(Z_(n′))₂ (12) at 14.35 minutes, PK2PYR(Z_(n′))₂(13) at 14.53 minutes.

HPLC Preparative:

Column: Water Xterra C18 prep MS Column 19×50 mm, 5 μm. Wavelength:220/280 nm. Flow rate: 8 ml/min. Solvent A=0.1% Trifluoroacetic acid.Solvent B=100% Acetonitrile. Temperature: 30° C. Gradient: 10-100% B, 80minutes. Pure fractions of PK1PYR(Z_(n′))₂ and PK2PYR(Z_(n′))₂ (13) werecollected and freeze dried separately to afford PK1PYR(Z_(n′))₂ (12) 49mg, and PK2PYR(Z_(n′))₂ (13) 42 mg, respectively.

PK1PYR(Z_(n′))₂ (12) MS indicated ESI +ve 10V 1331.5 ion. ¹H-NMR (D₂O) δ(ppm): 8.00 (2H, s, aromatic para H×2), 6.00 (2H, d, H-3×2), 5.20-5.50(4H, m, H-7×2, H-8×2), 4.35-4.90 (8H, m, H-4×2, H-5×2, H-6×2, H-9×2),4.15 (2H, dd, H-9′×2), 3.80 (6H, s, COOCH₃×2), 3.25-3.45 (4H, m,OCONHCH₂×2), 2.90-3.20 (4H, m, NHCH₂×2), 1.95 (6H, s, CH₃CO×2),1.20-1.70 (28H, m, (CH₂)₇×2).

PK2PYR(Z_(n′))₂ (13) MS indicated ESI +ve 10V 1331.5 ion. ¹H-NMR (D₂O) δ(ppm) 8.35 (1H, s, aromatic H×1), 7.50 (1H, s, aromatic H×1), 6.00 (2H,d, H-3×2), 5.20-5.50 (4H, m, H-7×2, H-8×2), 4.35-4.90 (8H, m, H-4×2,H-5×2, H-6×2, H-9×2), 4.15 (2H, dd, H-9′×2), 3.80 (6H, s, COOCH₃×2),3.25-3.45 (4H, m, OCONHCH₂×2), 2.90-3.20 (4H, m, NHCH₂×2), 1.95 (6H,CH₃CO×2), 1.20-1.70 (28H, m, (CH₂)₇×2).

Example 13: Preparation of Acidic/Anionic Group D-Asp(NHCH₂SO₃H)₂

Synthesis is detailed in Scheme 4 below. The following compoundreference numbers relate to Scheme 4.

Boc-D-Aspatic acid (14) (233 mg, 1 mmole) and HBTU[O-(benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate] (758 mg, 2 mmoles) were dissolved in DMF (10 ml).To this solution was added DIPEA (diisopropylethylamine) (349 μl, 2mmoles). The solution was stirred at room temperature for 10 minutes,then combined with a solution of sodium aminomethanesulfonate (332 mg,2.5 mmoles) in DMF (3 ml). The resulting solution was allowed to stir atroom temperature overnight. The reaction mixture was vacuum evaporatedto afford a pale orange solid. It was dissolved in water (20 ml), washedwith ethyl acetate (20 ml×3), the aqueous phase was vacuum evaporated toremove organic solvent, then freeze dried to afford crude product (15)310 mg as a white solid which was subjected to HPLC separation andpurification.

HPLC Analytical: Pepl Gradient

Column: Gemini C18 column 100A 5 μm 150×30 min. Wavelength: 220 nm. Flowrate: 0.7 ml/min. Solvent A=0.1% Trifluoroacetic acid. Solvent B=100%Acetonitrile. Temperature: 30° C. Gradient: 0-50% B, 15 minutes.Retention time: Compound (15) as free acid at 4.669 minutes MS ESI −ve418 (M−1), solvent peak at 1.658 minutes, aminomethanesulfonic acid at2.722 minutes, HBTU substance at 5.520 minutes, mono Boc-D-Asp-NHCH₂SO₃Hat 5.802 minutes.

HPLC Preparative

Column: Germini AXIA C18 column, 5 μm 50×21.2 mm Wavelength: 220 min.Flow rate: 8 ml/min; Solvent A=0.1% Trifluoroacetic acid; Solvent B=100%Acetonitrile; Temperature: 30° C. Gradient: 0-100° % B, 100 minutes.

Fraction containing (15) were collected and pooled together, then freezedried to afford pure product (15) as free acid. MS ESI −ve 418 (M−1).Compound (15) as free acid (200 mg, 0.477 mmoles) was dissolved intrifluoroacetic acid (10 ml) containing Anisole (1 ml). The solution wasstirred at room temperature for one hour, then was vacuum evaporated todryness. The residue was triturated in ether (20 ml×2), filtered off.The solid was air dried, then redissolved in water, freeze dried toafford product (16) 106 mg as a white solid. MS ESI −ve 318 (M−1). Theproduct was dissolved in water (10 ml), then neutralized with twoequivalent of sodium hydroxide, freeze dried to give disodium salt of(16) as a white solid.

Example 14: Preparation of Compound MD185,PK2PYR(Z_(n))₂[(D-Asp)(NHCH₂SO₃)₂]₂

Synthesis is detailed in Scheme 5 below. The following compoundreference numbers relate to Scheme 5.

To a solution of PK2PYR(Z_(n′))₂ (13) (30 mg, 22.56 μmoles) in dry DMF(5 ml) were added HATU [O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate] (17.14 mg, 45.11 μmoles) and DIPEA (7.9 μl, 45.31μmoles). The resulting solution was stirred at room temperature for 10minutes, then was added disodium salt of (16) [D-Asp(NHCH₂SO₃Na)₂] (33mg, 91.16 μmoles) in DMF (5 ml). The reaction mixture was stirred atroom temperature for 18 hrs, then it was subjected to HPLC separationand purification.

HPLC Analytical: Pep 1 Gradient

Column: Gemini C18 column 100A 5 μm 150×3.00 mm; Wavelength: 220/280 nm;Flow rate: 0.7 ml/min; Solvent A=0.1% Trifluoroacetic acid; SolventB=100% Acetonitrile; Temperature: 30° C.; Gradient: 0-50% B, 15 minutes;Retention time: The product (17) as free acid at 14.270 minutes. MS ESI+ve +30V 967.5. MS ESI −ve, −25 v 965.4. The other peaks were asfollows: the starting material PK2PYR(Z_(n′)) (13) at 11.7 minutes,PK2PYR(Z_(n′))₂-D-Asp(NHCH₂SO₃H)₂ at 13.6 minutes.

HPLC Preparative

Column: Water Xterra prep MS C18 column 19×50 mm 5 μm; Wavelength:220/280 nm; Flow rate: 8 ml/min; Solvent A=0.1% Trifluoroacetic acid;Solvent B=100% Acetonitrile; Temperature: 30° C.; Gradient: 0-100% B 100minutes

Product elutes in reverse profile to Gemini analytical. Elution profilebetween 25-35% acetonitrile. Fractions sampled and tubes with greaterthan 90% purity were pooled and other material was separated on theGemini AXIA column.

Product (17) was freeze dried to afford a white fluffy powder. MS ESI−ve −25 v 965.4, indicated the MW of 1932. Compound (17) PK2PYR(Z_(n′))₂[D-Asp(NHCH₂SO₃H)₂]₂ (8 mg, 4.14 μmoles) was dissolved in50% methanol aqueous solution (2 ml), then added triethylamine (40 μl,287.5 μmoles). The resulting solution was stirred at room temperature.Reaction was monitored by analytical HPLC for peak at 10.848 minutes.After 2 hrs, the reaction mixture was acidified with acetic acid (60 μl,1049 μmoles) and diluted to 10 ml with water. The solution was purifiedon the Kinetex column. Pure fractions were combined and freeze dried toafford product (18) PK2 PYR(Z_(n))₂[D-Asp(NHCH₂SO₃H)₂]₂ 6 mg as whitepowder.

HPLC retention time 10.695 min. MS ESI −ve −30V 925.6.

In order to remove residue of TFA in compound, the pure material wasdissolved in 60 ml 4 mM HCl and freeze dried, then dissolved in 60 ml1mMHCl and freeze dried, finally freeze dried from 60 ml water. Yield.5.6 mg (18) MD185 MW1852.

Example 15: Preparation of Anchor Compounds Z_(0′)

Synthesis is detailed in Scheme 6 below. Following compound referencenumbers and synthetic step references relate to Scheme 6.

According to the procedure (H) and (I) described at Example 11, reactionof compound (8) with p-nitrophenyl chloroformate, thenN-Boc-1,8-diaminooctane, after chromatography, afforded compound (19) asa white solid. MS 843 (M+1) Compound (19) was treated withtrifluoroacetic acid (TFA), after work-up to give compound (20) Z_(o′)as a white foam of TFA₃Z_(0′) salt MS 543 (M+1) [MW of Z_(o′)=542,TFA₃Z_(0′)=884].

Example 16: Preparation of Anchor-Backbone Compounds PK1 TCA(Z_(0′))₂and PK2 TCA(Z_(0′))₂

Synthesis is detailed in Scheme 7 below. The following compoundreference numbers relate to Scheme 7.

To a solution of TCA (21) (3.085 mg, 17.52 μmoles) in DMF (309 μl) wereadded HATU (13.32 mg, 35.03 μmoles) and DIPEA (6.5 μl, 37.32 μmoles).The whole mixture was stirred at room temperature for 10 minutes, thenwas added dropwise a solution of Z_(o′), (20) (30.97 mg, 35.03 μmoles)and DIPEA (18.44 μl, 105.9 μmoles) in DMF (600 μl). The reaction mixturewas allowed to stir overnight at room temperature. The resulting mixturewas subjected to HPLC separation and purification.

HPLC Analytical: Pepl Gradient

Column: Phenomenex C18 5 μl 110A 150×3 mm Wavelength: 220/280 nm; Flowrate: 0.7 ml/min; Solvent A=0.1% Trifluoroacetic acid; Solvent B=100%Acetonitrile; Temperature: 30° C.; Gradient: 100-50% B 15 minutesRetention time: PK1 TCA(Z_(o′))₂ (22) 11.100 min and PK2 TCA(Z_(o′))₂(23) 11.156 min.

PK1 TCA(Z_(o′))₂ (22) ¹H-NMR (D₂O) δ ppm at 2.5 ppm (AB symmetric dd,4H, C_(a)H₂═C_(b)H₂) indicated its symmetric structure. MS 1225.4 (MW1224).

PK2 TCA(Z_(o′))₂ (23) ¹H-NMR (D₂O) δ ppm at 2.35-2.75 ppm (asymmetric,dd, 4H, (C_(a)H₂≠C_(b)H₂) indicated its asymmetric structure. MS 1225.4(MW 1224).

Preparative HPLC

The solution was acidified with a small amount of 1M acetic acid anddiluted to 25 ml in water. This was filtered through 0.45 μm syringefilter and pumped onto a Water Xterra prep MS C18 column 19×50 mm at 20%to 80% buffer A. Flow rate 8 ml/min gradient 0-100% B 100 minutes,wavelength 210/280 nm. Elution Profile Void DMF/DIPEA/HATU

The PK1 TCA(Z_(o′))₂ and PK2 TCA(Z_(o′))₂ were further purified andseparated on the Phenomenex Gemini 5 μm C18 110A Axia 50×21.2 mm columnin 0.1% TFA buffer.

PK1/PK2 ratio is about 1:2, both MS ESI +30V 1225.4.

Example 17: Preparation of Acidic Anionic Group [D-Cya-SO₃H]₇OH

Synthesis is detailed in Scheme 8 below. The following compoundreference numbers relate to Scheme 8.

As detailed in Scheme 8, solid phase synthesis was applied to prepare[D-Cys-SH]₇—OH (33), then oxidation of the polycysteine (33) provided[D-CyaSO₃H]₇—OH (34).

Fmoc-D-Cys(Trt)-OH (24) (2.93 g, 5 mmoles) was dissolved in drydichloromethane (DCM) (35 ml) in a 50 ml Falcon tube, then was added2-chlorotrityl chloride resin (25) (5 g), shaken vigorously,diisopropylethylamine (DIPEA) (3.5 ml, 20 mmoles) was added. After 5min, another portion of DIPEA (1.3 ml, 7.5 mmoles) was added. The wholemixture was shaken for a further 4 hrs. To endcap any remaining reactive2-chlorotrityl chloride, methanol was added (4 ml). The mixture wasshaken for further 30 minutes. The resin was washed successively withDMF (20 ml×3). DCM (20 ml×3), methanol (20 ml×3), then vacuum driedovernight. Yield indicated loading of 0.57 mM/g (26).

For removal of Fmoc group from resin, the resin (26) was pre swollen inDCM (35 ml), then was added 50% piperidine in DMF (30 ml). The wholemixture was placed on a rotator for 30 min. This was repeated with afresh solution of 50% piperidine in DMF (30 ml) for 60 min. Resin wasthen washed successively with DMF (20 ml×2), DCM (20 ml×2), DMF (20ml×3). A positive ninhydrin test was presented. The resin (27) was readyfor coupling.

To a solution of Fmoc-D-Cys(Trt)-OH (24) (1758 mg, 3 mmoles) in DMF (6ml) were added HBTU[O-(Benzotriazol-1-yl)-N,N,N′,N′-bis(tetramethylene)uroniumhexafluorophosphate] (1140 mg, 3 mmoles) and DIPEA (523 μl, 3 mmoles).The mixture was stirred at room temperature for 10 minutes, then wasadded resin (27) (2.5 g, 0.57 mM/g, 1.5 mmoles). The reaction mixturewas allowed to agitate for 24 hrs. Ninhydrin test negative, the resinwas washed with DMF (20 ml×3), then treated with 5% acetic anhydride, 1%DIPEA in DMF (20 ml) for 30 minutes. The resin (28) was then washed withDMF (20 ml×3). DCM (20 ml×3), methanol (20 ml×3). The resin (28) was preswollen in DCM, and was added 30% piperidine/N-methylpyrollidone in DMF(20 ml). The whole mixture was placed on a rotator for 30 minutes, thenwith fresh solution of 30% piperidine and N-methylpyrollidone for afurther 60 minutes, after washing with DMF (20 ml×2), DCM (20 ml×2), DMF(20 ml×2) to afford resin (29).

The resin (29) was further coupled for 24 hrs. with (24) (1758 mg, 3mmoles) in DMF (6 ml) preactivated for 10 minutes with HBTU (1140 mg, 3mmoles) and DIPEA (523 μl, 3 mmoles), after work-up to afford resin(30). Half of which was then following the procedure: deprotected bypiperidine/methylpyrollidone, recoupled with (24) preactivated withHBTU/DIPEA, to repeat this procedure for 4 times to produce resin (31).

Resin (31) was washed with DCM (20 ml×5), then was placed in a 50 mlFalcon tube, added 40% acetic acid in DCM (35 ml), on a rotator at roomtemperature was shaken for 6 hrs. The resin suspension was filtered, thefiltrate was vacuum evaporated to a pale yellow foam. The residue waswashed with water (100 ml×3), after dried to afford a white solid (32)[D-Cys(Trt)]₇-OH.

(32) was stirred in a solution of TFA (20 ml), Triisopropylsilane (1 ml)and water (0.5 ml) for 3 hrs. The resulting suspension was filtered. Thefiltrate was evaporated to an oily substance which was then trituratedwith ether (20 ml×4) to give a white solid. It was stirred in 50%acetonitrile aqueous solution (100 ml). The material was sonicated toform a white suspension, which was freeze dried to afford a crudeproduct of (33) [D-Cys-SH]₇—OH (380 mg).

HPLC Analytical

Column: Phenomenex Gemini 5 μm C18 110A 150×3.00 mm Solvent A=0.1%Trifluoroacetic acid, Solvent B=100% Acetonitrile, Flow rate: 0.7ml/min, Wavelength: 210/280 nm, Temperature: 30° C., Gradient: 0-50% B15 minutes, Retention time: 9.356 minutes.

MS Cone +25V Major peak 740 Cone −25V Major peak 738 Indicated MW of(33) is 739 [D-Cys-SH]₇—OH

Compound (33) (10 mg, 13.53 μmoles) was added to a performic acidsolution (1 ml) prechilled in an ice-salt bath. The whole mixture wasstirred in the ice bath for 1 hr, then diluted with 100 ml cold water,freeze dried to afford a pale yellow form (34). This material waspurified on a Phenomenex Kinetex 5 μm XB-C18 100A column using isocratic0.1% TFA as eluent to afford compound (34) [D-Cyst-SO₃H]₇—OH MW 1075, MSCone +50V 1076 (M+1) as a colourless powder.

Performic acid was prepared by mixing Formic acid (8.74 ml), water (0.96ml) and 30% hydrogen peroxide (1 ml), and allowing the mixture to standat room temperature for 30 minutes in a stopped flask. This peroxideshould be freshly made before use.

Example 18: Preparation of Compound MD345, PK2TCA(Z₀)₂-[D-Cyst-SO₃H]₇—OH

Synthesis is detailed in Scheme 9 below. The following compoundreference numbers relate to Scheme 9.

To a solution of PK2 TCA(Z_(0′))₂ (23) (4 mg, 3.268 moles) in anhydrousDMF (2 ml) were added HATU (1.242 mg, 3.268 μmoles) and 2% DIPEA in DMF(30 μl, 3.44 μmoles). The mixture was stirred at room temperature for 10minutes, then combined with a solution of (34), [D-Cyst-SO₃H]₇—OH (7.03mg, 6.54 moles) and DIPEA (9.11 μl, 52.32 μmoles), in DMF (3 ml). Thewhole reaction mixture was stirred at room temperature overnight. Themixture was diluted to 15 ml with water/methanol 1/1, then subjected toHPLC separation and purification.

HPLC Preparative:

Column: Phenomenex Kinetex 5 μm XB-C18 50×21.2 mm; Wavelength: 220/280nm; Flow rate: 8 ml/min; Solvent A=0.1% Trifluoroacetic acid (TFA);Solvent B=100% Acetonitrile; Temperature: 30° C.; Gradient: 0-100% B, 80minutes. Fractions containing (35) free acid (B═H) were combined andfreeze dried to afford compound (35) free acid (B═H) 4 mg as a whitepowder. MS ESI +ve Cone 35V 1142.1 2+ion.

Compound (35) free acid (B═H), PK2 TCA(Z_(0′))₂-[D-Cyst-SO₃H]₇—OH (4 mg,1.754 μmoles) was dissolved in 50% methanol aqueous solution (800 μl)containing triethylamine (TEA) (20 μl, 146 μmoles). The mixture wasstirred at room temperature, monitored by HPLC. It was worked up after 3hrs. The solution was purified on the Kinetex column. Pure fractions ata retention time of 11.254 min were combined and freeze dried to affordcompound (36) 2 mg as a white powder. MS ESI +ve Cone 40V 1102.2 2+ion.

In order to remove the residue of TFA in the compound, compound (36) wasdissolved in 4 mM HCl (30 ml) and freeze dried. Then it was dissolved in1 mM HCl (30 ml) and freeze dried. Finally it was freeze dried fromwater (30 ml) to afford product (36) 1.1 mg, MD345 PK2TCA(Z₀)₂[D-Cyst-SO₃H]₇—OH MW2201 [MS 2202 (M+1)].

Example 19: Preparation of Anchor Compound Z′

Synthesis is detailed in Scheme 10 below. Following compound referencenumbers and synthetic step references relate to Scheme 10.

According to the procedure (H) and (I) described in Example 11, reactionof compound (8) with p-nitrophenyl chloroformate, thenN-Boc-1,6-diaminohexane, after chromatography, to afford compound (37)as a white solid, MS 815 (M+1). Compound (37) was treated withtrifluoroacetic acid (TFA) and anisole, after work up to give compound(38) Z′ as a white form of TFA salt. MS 515 (M+1). [MW of Z′=514.TFA₃Z′=856].

Example 20: Preparation of Anchor-Backbone Compound CHTCA(Z′)₂

Synthesis is detailed in Scheme 11 below. Following compound referencenumbers relate to Scheme 11.

To a solution of CHTCA(39) (4.4 mg, 20 μmoles) in DMF (300 μl) was addedHATU (15.2 mg, 40 μmoles) and DIPEA (7.4 μl, 42.4 μmoles). The wholemixture was stirred at room temperature for 10 minutes, then was addeddropwise a solution of Z′ (38) (34.24 mg, 40 μmoles) and DIPEA (21.06μl, 120.9 μmoles) in DMF (700 μl). The reaction mixture was allowed tostir overnight at room temperature. The resulting mixture was dilutedwith 1M HAc (50 μl) and water (25 ml). This was filtered through 0.45 μmsyringe filter. The filtrate was subjected to HPLC separation andpurification.

Column: Water Xterra prep MS C18 column 19×50 mm; Wavelength: 210/280nm; Flow rate: 8 ml/min, Solvent A=0.1% TFA Solvent B=100% Acetonitrile;Temperature: 30° C.; Gradient: 0-100% B 100 minutes; Elution ProfileVoid DMF/DIPEA/HATU.

The fractions containing compound (40) were further purified on thePhenomenex Gemini 5 μm C18 110A AXIA 50×21.2 mm column in 0.1% TFAbuffer. The fraction was freeze dried to afford compound (40)CHTCA(Z′)₂, 15 mg as a white foam. MS ESI +ve, +30 v, 1209 indicated theMW of 1208.

Example 20: Preparation of Anchor Compound S₆

Synthesis is detailed in Scheme 12 below. The following compoundreference numbers and synthetic step references relate to Scheme 12.

Step A) Compound (1) was stirred in anhydrous methanol (100 ml) at roomtemperature for 48 hrs, the mixture was filtered off. The filtrate wasvacuum evaporated to dryness to yield compound (41), 1.25 g (6.037mmoles, 93% Yd.) as a white solid. MS 324 (M+1).

Step B) Compound (41) (1.95 g, 6.037 mmoles) was stirred in acetylchloride (20 ml, 281 mmoles) at room temperature for 3 days, then vacuumevaporated to dryness to afford compound (42) 3.06 g (6.01 mmoles, 99%Yd.). MS 510.5 (M+1).

Step C) The residue (42) (3.06 g, 6.01 mmoles) was stirred indichloromethane (DCM) (35 ml), then added KSAc (3.57 g, 31.26 mmoles).The reaction mixture was stirred under argon at room temperature for 40hrs, diluted with dichloromethane (50 ml), then was partitioned betweendichloromethane and water (50 ml). The organic layer was washed with 5%NaCl solution (22 ml×2), and dried over anhydrous Na₂SO₄ overnight,filtered off. The filtrate was evaporated to dryness to yield (43) 2.93g (5.34 mmoles, 88.8% Yd.) as an off white foam. MS 550 (M+1).

Step D) Compound (43) (200 mg, 0.364 mmoles), and Boc-aminohexanebromide (107.4 mg, 0.385 mmoles) were stirred in DMF (2 ml), into whichwas added diethylamine (0.81 ml, 7.83 mmoles). The whole mixture wasstirred under argon at 25° C. for 2.5 hrs. The reaction mixture waspartitioned between ethyl acetate (EA) (80 ml) and saturated NaClsolution. The organic layer was washed with saturated NaCl solution (15ml×3), water (10 ml×3). The organic layer was dried over Na₂SO₄,filtered, the filtrate was vacuum evaporated to dryness. The residue wassubjected to silica gel column chromatography (Toluene:Acetone=2:1). Thefractions containing compound (44) were combined and vacuum evaporatedto dryness to afford compound (44) 98 mg (0.139 mmoles, 38% Yd.) as awhite foam. MS 706 (M+1).

Step E) Compound (44) (95 mg, 0.135 mmoles) was dissolved in anhydrousmethanol (1 ml). To this solution was added sodium methoxide (0.16 mg,6.94 μmoles) under argon, the reaction mixture was stirred at roomtemperature for 2.5 hrs, then was adjusted with Dowex50×8(H⁺) resin topH 6.0-6.5, filtered off. The filtrate was evaporated under vacuum todryness to afford compound (45) 71 mg (0.132 mmoles, 97.9% Yd.) as awhite foam. MS 538 (M+1)

Step F) Compound (45) (60 mg, 0.109 mmoles) was stirred under argon in0.2N NaOH (2 ml, 0.4 mmoles) at room temperature for 2.5 hrs. Thesolution was adjusted to pH 3-4 with Dowex50×8(H⁺) resin and filteredoff. The filtrate was freeze dried to afford compound (46) 56 mg (0.107mmoles, 98% Yd.) as a white foam. MS 524 (M+1).

Step G) Compound (46) (50 mg, 0.0956 mmoles) was dissolved in a mixtureof trifluoroacetic acid (TFA) (2 ml, 25.96 mmoles) and methylphenylether(anisole) (0.2 ml, 1.84 mmoles) in dichloromethane (2 ml). The wholemixture was stirred at 25° C. for 2.5 hrs, then it was vacuum evaporatedat 35° C. for 2 hrs. The residue was washed in hexane (10 ml×2) at roomtemperature overnight, the hexane was decanted. The residue was stirredin ether (10 ml×2) at room temperature, then ether was removed. Theresidue was dissolved in water (1 ml), and freeze dried to affordcompound (47), as a white foam of S₆.TFA salt, 51 mg (0.0949 mmoles, 99%Yd.). MS 424 (M+1) [The MW of S₆=423, S₆.TFA salt=537].

Example 21: Preparation of Compound MD012, CHTCA(Z)₂(S₆)

Synthesis is detailed in Scheme 13 below. The following compoundreference numbers and synthetic step references relate to Scheme 13.

To a solution of CHTCA(Z′)₂ (40) (4 mg, 3.3 μmoles) in DMF (100 μl) wasadded HATU (1.254 mg, 3.3 μmoles) and DIPEA (0.6 μl, 3.44 μmoles). Themixture was stirred at room temperature for 10 minutes, then was added asolution of S₆.TFA salt (47) (1.8 mg, 3.35 μmoles) and DIPEA (1.20 μl,6.89 μmoles) in DMF (100 μl). The reaction mixture was stirred at roomtemperature overnight. The resulting mixture was diluted with 1M HAc (5μl) and water (2 ml), then it was filtered through 0.45 μm syringefilter. The filtrate was subjected to HPLC separation and purification.

Column: Phenomenex Gemini AXIA 5 μm, C18 110A 50×21.2 mm; Wavelength:210/280 nm; Flow rate: 8 ml/min; Solvent A=0.1% Trifluoroacretic acid inMilliq water; Solvent B=100% Acetonitrile; Gradient: 0-100% B, 100minutes, linear; Temperature: 30° C.

The fractions containing compound (48) were collected and freeze driedto afford compound (48) CHTCA(Z′)₂(S₆) 2.4 mg, as a white foam. MS ESI+30 v 1615 indicated MW of 1614.

Compound (48) (2.4 mg, 1.486 μmoles) was stirred in 50% methanol aqueoussolution (90 μl) containing triethylamine (6.6 μl) at room temperaturefor 5 hrs, then vacuum evaporated to dryness. The residue wasredissolved in water (100 μl), freeze dried to afford the product (49)CHTCA(Z)₂(S₆), 2 mg (1.303 mmoles 87.7% Yd.) as a white foam. MS ESI +30v 1535.7, indicated the MW of 1534.7

Example 22: Preparation of Anchor-Backbone Compound CHTCA(Z_(0′))₂

Synthesis is detailed in Scheme 14 below. The following compoundreference numbers relate to Scheme 14.

The preparation of anchor compound (20) Z_(0′) is detailed at Example15. Anchor-backbone compound (50) CHTCA(Z_(0′))₂ was prepared accordingto the procedure detailed in Example 20. The Anchor-backbone compound(50) CHTCA(Z_(0′))₂ was obtained as a white solid. MS ESI +ve, +30 v1265 indicated the MW of 1264

Example 23: Preparation of Acidic/Anionic GroupD-Asp(D-Cya)-D-Asp(D-Cya)-D-Asp(D-Cya)-OH

Synthesis is detailed in Scheme 15 below. The following compoundreference numbers (A) to (1) and synthetic step references relate toScheme 15.

Boc-D-Cysteine(Acm)methyl ester, [Boc-D-Cys(Acm)-OMe] (1.532 g, 5mmoles) was dissolved in trifluoroacetic acid (TFA) (10 ml) containinganisole (1 ml), and stirred at room temperature for 30 minutes. Thereaction mixture was then evaporated under vacuum to remove TFA toafford an oily substance, which was triturated in ether (100 ml×2) thenredissolved in 20% acetonitrile aqueous solution (100 ml), and freezedried for 72 hrs to give compound TFA.D-Cys(Acm)-OMe, 1.60 g. HPLC/MSindicated the MW of 206.35 (as a free base).

Fmoc-D-Aspartic-α-acid-β-O-t-butyl ester, [Fmoc-D-Asp-Obut], (1.646 g, 4mmoles) was activated with HBTU (MW 379.25) (1.517 g, 4 mmoles) andDIPEA (MW 129.25) (0.517 g, 4 mmoles) in DMF (20 ml) at room temperaturefor 10 minutes, then combined with a solution of TFA.D-Cys(Acm)-OMe(1.47 g, 4.2 mmoles) and DIPEA (0.542 g, 4.2 mmoles) in DMF (10 ml). Thewhole reaction mixture was allowed to stir for 16 hrs at roomtemperature. The reaction was monitored by HPLC. The mixture wasevaporated under vacuum to remove DMF. Then water (5×70 ml) was added tothe residue to wash away residual DMF and D-Cys(Acm)OMe to give an offwhite solid, which was then vacuum dried to yield compound (A),Fmoc-D-Asp[D-Cys(Acm)-OMe]-O-But as a white solid. HPLC/MS indicated MWof 599.7.

Compound (A) (1.2 g, 2 mmoles) was treated with 20% piperidine inacetonitrile (20 ml) for 30 minutes at room temperature, then filteredoff. The filtrate was evaporated under vacuum to afford compound (B),D-Asp[D-Cys(Acm)-OMe]-OBut. HPLC/MS indicated MW of 377.4.

Compound (A) (599.7 mg, 1 mmole) was treated with TFA (6 ml) for 1 hr atroom temperature. Then it was vacuum evaporated to dryness. The residuewas triturated in ether (50 ml×3), then dissolved in 30% acetonitrileaqueous solution (100 ml), and freeze dried to afford compound (C),Fmoc-D-Asp[D-Cys(Acm)-OMe]-OH as a white solid. HPLC/MS indicated MW of542.7.

Compound (C), Fmoc-D-Asp[D-Cys(Acm)-OMe]-OH (434 mg, 0.8 mmoles) wasactivated with HBTU (303.4 mg, 0.8 mmole) and DIPEA (103.4 mg, 0.8mmole) in DMF (8 ml) at room temperature for 10 minutes, then it wasadded compound (B), D-Asp[D-Cys(Acm)-OMe]-OBut (9332 mg, 0.88 mmoles).The reaction mixture was stirred at room temperature for 16 hrs. The DMFwas evaporated under vacuum and the residual DMF was removed by washingthe white solid material carefully with water. Then the white solid wasdried under vacuum for 24 hrs to afford compound (D),Fmoc-D-Asp[D-Cys(Acm)-OMe]-D-Asp[D-Cys(Acm)-OMe]-OBut, as a white solid.HPLC/MS indicated the MW of 902.19. Compound (D) was treated with TFA(16 ml) at room temperature for 1 hr to remove t-Butyl ester. The TFAwas vacuum evaporated to dryness. The residue was washed with water toremove residual TFA and the solid was vacuum dried for 72 hrs to yieldFmoc-D-Asp[D-Cys(Acm)-OMe]-D-Asp[D-Cys(Acm)-OMe]-OH. HPLC/MS indicatedthe MW of 846.

Fmoc-D-Asp[D-Cys(Acm)-OMe]-D-Asp[D-Cys(Acm)-OMe]-OH (423 mg, 0.5 mmol)was activated with HBTU (189.5 mg, 0.5 mmole) and DIPEA (64.63 mg, 0.5mmole) in DMF (5 ml) at room temperature for 10 minutes, then was addedcompound (B), D-Asp[D-Cys(Acm)-OMe]-OBut (317 mg, 0.84 mmole). The wholemixture was stirred at room temperature for 16 hrs. The reaction waschecked by HPLC for Completion, and the DMF was removed under vacuum.The white solid obtained was carefully washed with cold water then driedunder vacuum for 48 hrs to afford Compound (E),Fmoc-D-Asp[D-Cys(Acm)-OMe]-D-Asp[D-Cys(Acm)-OMe]-D-Asp[D-Cys(Acm)-OMe]-OBut,MW 1206, which was further purified by HPLC.

Analytical HPLC of this material indicated a purity of 70%. The materialwas dissolved in 50% methanol/water and filtered through 0.2 μm filterfor preparative HPLC.

Preparative HPLC

Column: Waters Xterra C18 column 5 μm 19×50 mm in batches of 40 mg;Wavelength: 220/280 nm; Flow rate: 8 ml/min; Solvent A=0.1%Trifluoroacetic acid; Solvent B=100%, Acetonitrile; Gradient: 10-100% B,100 minutes; Temperature: 30° C.

Compound (E),Fmoc-D-Asp[D-Cys(Acm)-OMe]-D-Asp[D-Cys(Acm)-OMe]-D-Asp[D-Cys(Acm)-OMe]-OButwas then treated with TFA (10 ml) for 1 hr at room temperature andvacuum evaporated to dryness. The residue was dissolved in 40%acetonitrile aqueous solution (100 ml), and freeze dried to affordcompound (F),Fmoc-D-Asp[D-Cys(Acm)-OMe]-D-Asp[D-Cys(Acm)-OMe]-D-Asp[D-Cys(Acm)-OMe]-OH,as a white solid. HPLC/MS indicated the MW of 1150.27.

Compound (F),Fmoc-D-Asp[D-Cys(Acm)-OMe]-D-Asp[D-Cys(Acm)-OMe]-D-Asp[D-Cys(Acm)-OMe]-OH(200 mg, 0.1738 mmoles) was treated with 20% piperidine in acetonitrile(10 ml) for 30 minutes with stirred at room temperature and subsequentlyfiltered off. The filtrate was vacuum evaporated. The residue wasstirred in 50% water/methanol (20 ml) containing triethylamine (500 μl)at room temperature for 6 hrs, monitored by HPLC to indicate thecompletion of the reaction. This mixture was vacuum evaporated todryness to afford compound (G),D-Asp[D-Cys(Acm)-OH]-D-Asp[D-Cys(Acm)-OH]-D-Asp[D-Cys(Acm)-OH]—OH.HPLC/MS indicated the MW of 885.9.

Analytical HPLC indicated 50% purity.

Compound (G) was further purified by preparative HPLC.

Column: Phenomenex Gemini Axia 110A 5 μm 50×21.2 mm C18 HPLC columnWavelength: 210/280 nm; Flow rate: 8 ml/min; Solvent A=0.1% TFA; SolventB=100% Acetonitrile; Gradient: 0-100% B, 100 s minutes; Temperature: 30°C.

Sample (100 mg) was dissolved in 10% methanol aqueous solution,sonicated, and filtered through a 0.2 μm filter. 20 mg/batches werepumped into the column with a gradient run. Fractions were monitored byanalytical RF-HPLC using a Phenomenex Gemini C18 5 μm column. Flow rate0.7 ml/min; Wavelength 210/280 nm; Gradient 0-50% B 15 minutes; SolventA=0.1% TFA; Solvent B=100% Acetonitrile.

Fractions with the correct MS great than 90% purity were pooled andfreeze dried to afford purified compound (G).

Compound (G), D-Asp[D-Cys(Acm)-OH]-D-Asp-[D-Cys(Acm)-OH]-D-Asp[D-Cys(Acm)-OH]—OH, (21.8 mg,0.0246 mmoles) was dissolved in TFA (10 ml) containing anisole (200 μl),then was added silver trifluoromethanesulfonate (504 mg, 1.96 mmoles).The reaction mixture was stirred at room temperature for 1 hr. Then itwas vacuum evaporated to dryness. The residue was triturated in ether(40 ml×2), the ether washing were decanted. The residue was stirred in1M acetic acid (20 ml) containing dithiothreitol (403 mg, 2.62 mmoles)at 25° C. for 3 hrs. The suspension was centrifuged in 50 ml falcontubes for 10 minutes at 4000 rpm. The supernatant was carefullycollected, then purified by, HPLC and freeze dried to afford compound(H), D-Asp(D-Cys-SH,—OH)-D-Asp(D-Cys-SH,—OH)-D-Asp(D-Cys-SH,—OH)—OH,HPLC/MS indicated the MW of 672.3.

A mixture of 95% formic acid (7.36 ml), 30% H₂O₂ (0.8 ml) and H₂O (0.368ml) was left at room temperature for 30 minutes, then was stirred at −5°C. ice-bath, to which was added compound (H) (8 mg, 0.0119 mmoles) andstirred for 1 hr, then under vacuum for 10 minutes. The remainingsolution was then diluted with water to 200 ml and freeze dried toafford compound (I) 9.5 mg,D-Asp[D-Cya-SO₃H,—OH]-D-Asp[D-Cya-S₃OH,—OH]-D-Asp[D-Cya-SO₃H,—OH]—OH asa white solid, HPLC/MS indicated the MW of 816.78.

This compound was purified by desalting under the following conditions.

Column: Phenomenex Gemini Axia 110A 5 μm 50×21.2 mm C18 HPLC column;Wavelength: 210/280 nm; Flow rate: 8 ml/min; Solvent A=0.1% TFA; SolventB=100% Acetonitrile; Gradient: 0-100% B, 100 minutes Temperature: 30° C.The compound (I) elutes near the void.

This material was then freeze dried to produce a white powder as purecompound (I). Compound (I) was neutralized with triethylamine (TEA) toform a TEA salt (51) as follows:

-   *HBTU: O-1H-Benzotriazol-1-yl)-N,N,N′,N′-tetramethyl-uranium    hexafluorophosphate MW379.24-   TFA: Trifluoroacetic acid MW 114.02-   DIPEA: Diisopropylethylamine MW 129.25-   TEA; Triethylamine MW 101.19-   Acm: S-Acetamidomethyl as thio protecting group-   OBut: t-Butyl ester

Example 24: Preparation of Compound MD348,CHTCA(Z₀)₂-[D-Asp(D-Cyst)-D-Asp(D-Cyst)-D-Asp(D-Cyst)]-OH

Synthesis is detailed in Scheme 16 below. The following compoundreference numbers relate to Scheme 16.

Compound (50), CHTCA(Z_(0′))₂ from Example 22 (10.744 mg, 8.5 μmoles)was stirred with HATU (3.23 mg, 8.5 μmoles) and DIPEA (1.496 μl, 8.56μmoles) in DMF (5 ml) at room temperature for 10 minutes, then it wascombined with compound (51),D-Asp(D-Cya-SO₃B,—OB)-D-Asp-(D-Cya-SO₃B,—OB)-D-Asp(D-Cya-SO₃B,—OB)—OB,from Example 23 (13.2 mg, 8.67 μmoles) in DMF (5 ml). The whole mixturewas stirred at room temperature for 16 hrs. The resulting mixture wasmonitored by analytical HPLC/MS at retention time Peak 1, 12.903 min.44% MS ESI +ve, +20 v 1032.3 2+ion, ESI −ve, −40 v 1030.7 2+ion,indicated the MW of 2062 as compound (52) without base, that is,CHTCA(Z₀)₂[D-Asp(D-Cya-SO₃H,—OH)-D-Asp(D-Cya-SO₃H,—OH)-D-Asp-(D-Cyst-SO₃H,—OH)]—OH.

Analytical HPLC:

Column: Phenomenex Kinetex EVO 5 μm C18 100A 150×4.6 mm; Wavelength:210/280 nm; Flow rate: 0.7 ml/min; Solvent A=0.1% TFA; Solvent B=100%Acetonitrile; Gradient: 0-50% B, 15 minutes, linear; Temperature: 30° C.

The resulting mixture was acidified with citric acid, diluted to 20 mlwith water, and filtered through 0.2 μm filter, then subjected topreparative HPLC for separation and purification.

Preparative HPLC

Column: Phenomenex Gemini AXIA 5 μm C18 110A 50×21.2 mm; Wavelength:210/280 nm; Flow rate: 8 ml/min; Solvent A=0.1 TFA; Solvent B=100%Acetonitrile; Gradient: 0-100% B, 100 minutes, linear; Temperature: 30°C.

Major peak with retention time of 31.087 min was the product (52).Fractions containing product were collected and repurified by furtherHPLC as follows.

Column: Phenomenex Kinetex×B 5 μm C18 100A 50×21.2 mm; Wavelength:210/280 nm; Flow rate: 8 ml/min; Solvent A=0.1 TFA; Solvent B=100%Acetonitrile; Gradient: 5%-100% B, 80 min, linear; Temperature 30° C.

The purified compound (52) at retention time of 19.597 min wascompletely separated from the starting material at retention time of21.714 min.

Compound (52) (3.5 mg, 1.697 μmoles) was dissolved in 50% methanol/water(5 ml) containing triethylamine (TEA) (18 μl, 129 μmoles). It wasstirred at room temperature for 3 hrs. HPLC indicated the incompletionof the reaction, therefore, an additional 11 μl of TEA was added. Themixture was stirred for another 60 minutes by then HPLC indicated thecompletion of the reaction. The reaction solution was then neutralizedto pH 6 with 1M acetic acid. The solution was then diluted to 20 ml withwater. The mixture was subjected to HPLC.

Analytical HPLC

Column: Phenomenex Kinetex Evo 5 μm C18 100A 150×3 mm; Wavelength:210/280 nm Flow rate: 0.7 ml/min; Solvent A=20 mM NaHPO₄ pH 7.0 buffer;Solvent B=10 mM NaHPO₄ pH 7.0 buffer+50% Acetonitrile; Gradient: 0-100%B, 30 minutes, linear; Temperature: 30° C.

Preparative HPLC

Column: Phenomenex Kinetex XB 5 μm C18 100A 50×21.2 mm; Wavelength:210/280 nm Flow rate: 8 ml/min; Solvent A=0.1% TFA Solvent B=100%Acetonitrile Gradient: 0-100% B, 80 min, linear; Temperature: 30° C.

The product (53) was eluted at a retention time of 16.431 min.

Pure fractions were combined and freeze dried, then using HCl exchangeto remove the residual TFA. Freeze drying afforded the product (53)CHTCA(Z₀)₂[D-Asp(D-Cya)-D-Asp(D-Cya)-D-Asp(D-Cya]-OH at purity >99% as awhite solid. MS ESI cone −20 v, 990.5 2+ion indicated the MW of 1982.

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We claim:
 1. A compound of Formula (I):[A-L¹

_(n)B

L²-C]_(p)   Formula (I) or a pharmaceutically acceptable salt, solvate,ester or stereoisomer thereof, wherein: A at each occurrence is ananchoring group selected from the group consisting of zanamivir,laninamivir, oseltamivir, peramivir, and 2,3-difluoro sialic acids or anantibody binding domain which specifically binds neuraminidase; B is amultivalent backbone group selected from the group consisting ofoptionally substituted propane-1,2,3-tricarboxylate,prop-1-ene-1,2,3-tricarboxylate, cyclopropane-1,2,3-tricarboxylate,cyclohexane-1,3,5-tricarboxylate, benzene-1,3,5-tricarboxylate, methanetetracarboxylate, 1,2,3,4-butane tetracarboxylate,ethylene-1,1,2,2-tetracarboxylate, cyclohexane-1,2,4,5-tetracarboxylate,and benzene-1,2,4,5-tetracarboxylate; C at each occurrence is an anionicgroup of Formula VIII:

wherein R⁴ at each occurrence is independently selected from the groupconsisting of —COOH, — SO₃H, —NHCH₂SO₃H, —CONH-Cya, -CONN-Asp,—CONH-Asp(-NHCH₂SO₃H)_(w), and —CONH-Asp(-NHCH₂PO₃H)_(w), wherein w isan integer 1 or 2; R⁵ is selected from the group consisting of —OH and—NHCH₂SO₃H; u is an integer from 0 to 3; t is an integer from 0 to 3; ris an integer from 0 to 6; and v is an integer from 1 to 12; L¹ and L²at each occurrence are divalent linkers; n is 2 or 3; and p is 1 or 2.2. The compound of claim 1, wherein the anchoring group is selected fromthe group consisting of an antibody (Ab) or an antigen binding fragmentof an antibody, a single chain antibody fragment (scFV) and a singledomain antibody (dAb).
 3. The compound of claim 1 or claim 2, wherein L¹and L² at each occurrence are independently selected from the groupconsisting of a bond, optionally substituted C₁-C₁₂ alkylene, —C(O)—NR—,—O—C(O)O—NR—, —C═N—O—, —C═N—NR—, —SO₂—NR—, disulfide,—C(O)—NR—(CH₂)_(x)—NR—, —(CH₂)_(x)—NR—, —(CH₂)_(x)—(O—(CH₂)_(y)—)_(z)—,optionally substituted-C═N—O—, optionally substituted —C═N—NR-alkylene,optionally substituted alkylene sulfonamide, an optionally substitutedalkylene disulfide, wherein R is independently H or C₁-C₆ alkyl andwherein x, y and z are each integers independently selected from 0, 1,2, 3 and
 4. 4. The compound of any one of claims 1, 2, or 3, wherein Bis selected from the group consisting of optionally substitutedpropane-1,2,3-tricarboxylate, optionally substitutedcyclohexane-1,3,5-tricarboxylate, and optionally substitutedbenzene-1,2,4,5-tetracarboxylate.
 5. The compound of claim 1 selectedfrom the group consisting of:

wherein Z₀ is;

Z is

and Z_(n) is

or a pharmaceutically acceptable salt, solvate, ester or stereoisomerthereof.
 6. The compound of claim 1, wherein A is zanamivir.
 7. Thecompound of claim 1, wherein B is benzene-1,2,4,5-tetracarboxylate. 8.The compound of claim 1, wherein R⁴ at each occurrence is COOH and R⁵ is—OH.
 9. The compound of claim 1, wherein L² at each occurrence, is abond.
 10. The compound of claim 1, wherein [A-L¹] at each occurrence, isrepresented by a group of formula Z_(x) having the structure:

wherein x is an integer from 0-12.
 11. The compound of claim 10, whereinZ_(x) is a group of formula Z_(n):


12. The compound of claim 1 of structure:

wherein Z_(n) is

or a pharmaceutically acceptable salt, solvate, ester or stereoisomerthereof.
 13. The compound of claim 12, wherein the compound is an alkylester.
 14. A pharmaceutical composition comprising a compound ofclaim
 1. 15. The pharmaceutical composition of claim 14, wherein thecompound is a compound of claim
 5. 16. A method of treating orpreventing influenza viral infection, comprising administering to aperson in need thereof, a therapeutically effective amount of a compoundof claim
 1. 17. The method of claim 16 wherein the influenza viralinfection is influenza A, influenza B, avian flu, or a drug resistantstrain of influenza.
 18. The method of claim 16 wherein the infection isa respiratory tract infection or a systemic infection.
 19. The method ofclaim 16, wherein the compound is selected from

wherein Z_(n) is

or a pharmaceutically acceptable salt, solvate, ester or stereoisomerthereof.